Christoper D. Krause
- Courses1
- Reviews1
- School: Indian River State College
- Campus:
- Department: Biology
- Email address: Join to see
- Phone: Join to see
-
Location:
3209 Virginia Ave
Fort Pierce, FL - 34981 - Dates at Indian River State College: December 2017 - December 2017
- Office Hours: Join to see
Biography
Indian River State College - Biology
Teacher, Scientist, Consultant, Videographer/Photographer
Biotechnology
Christopher D.
Krause
Port Saint Lucie, Florida
Proven expertise in and consultation available for:
- confocal fluorescence spectroscopy
- real-time analysis of protein interactions and signal transduction in live cells
- (pedagogical/persuasive) scientific or technological graphics design
- mathematical modeling
- scientific writing
- DNA vector design, recombinant protein expression.
Team leader training:
- Ph.D. dissertation in structural and molecular immunology
- multi-faceted approach to understand interferon-gamma signaling and receptor structure
Accomplished writer:
- Principal author of ten manuscripts, four reviews, and two book chapters
Effective team player:
- Co-author of nine manuscripts, two reviews, two book chapters
Public speaker and Presenter:
- Presented oral/poster presentations at 12 international conferences and 10+ domestic conferences
"Out of the box", unconventional problem analysis and solutions:
- Developed computer models to simulate signal transduction, FRET
- Derived methodology to analyze complex fluorescence emission spectra
Managerial experience:
- Directed multiple distinct research projects
- mentored graduate students
- scientific and technical advisor, scientific advisory board member
- course director of college-level biology courses
Tutor, Teacher and Trainer of both individuals and groups:
- Taught microbiology, immunology, general biology, chemistry, anatomy & physiology
- tutored students and athletes in math, chemistry, and biology
Experience
CDK Videography
Founder and Principal Photographer/Videographer
Photographic expertise: UV/vis/infrared photography, dim-light photography, astrophotography, technical photograhpy
Videographic expertise: product demonstration (outdoor and indoor). underwater videography, lecture demonstration and business-oriented presentationMJ BIOTECH,INC.
Member, Scientific Advisory Board
content expert in lipophilic signal transduction, molecular immunology, and molecular physiology
BioMune (http://www.bio-mune.com/)
Chief Science Officer, Member - Board of Directors
Develop and evaluate plant-based therapeutics that promote activation of innate and adaptive immunity to combat cancer
Evaluate conventional and alternate sources of natural biotherapeutics
Investigate new modalities of therapy
Facilitate collaborations with partnering companies or investors
update website, investor documents, presentations, business plan
The SoundWell Corp.
Chief Research & Development Officer
Investigate physiological mechanism of beneficial effects of vibro-acoustic therapy (VAT)
Increase awareness of VAT within wellness and alternative medical treatment communities
Generate case histories of individuals using VAT
Improve technology implementing VAT
Developed pitch and device demonstration videos for company and webpage development
created Facebook page for companyIndian River State College
Master Instructor
Teach a variety of biology courses (general biology lecture and laboratory to biology majors, nursing students, and non-majors, microbiology lecture and laboratory to nursing students) to a variety of undergraduate collegiate levels and dual-enrollment high-school students
participate in the development of online and virtual learning models,
course director for general biology for non-majors
Education
Rutgers/UMDNJ - Robert Wood Johnson Medical School
PhD
Biochemistry
Research advisor - Dr. Sidney Pestka, MD (dec.) Research focused on interactions among interferon-gamma receptor chains and their relationships to apoptosis and antiviral activity. Research performed in partial fulfillment; Teaching assistant in (1) medical school Microbiology and Immunology Laboratory and (2) undergraduate introduction to biochemistry Teacher's assistant in New Brunswick (NJ) School District - assisted 7th and 8th grade science teachers with science experiments and effective teaching of science to middle school students. Additional responsibilities included regular oral and written presentations of research progress, collaborating with labmates, computer maintenance and organization and maintenance of restriction endonuclease library; Undergraduate School and Undergraduate ResearchUniversity of Delaware, Department of Chemistry and Biochemistry
Honors BS
Biochemistry
class rank 321/3382. Research advisor: Mahendra K. Jain (dec.) Research focused on kinetic characterization and HPLC purification of porcine pancreatic lipase and a bacterial lipase/acyltransferase. Research done for Senior Honors Thesis in partial Fulfillment of the Honors Degree Tutor to undergraduate students and student-athletes in chemistry, biochemistry, organic chemistry, precalculus, calculus, french
Publications
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Toward the Development of Real-Time Cellular Kinetic Assays to Characterize Inhibitors of Cytokine Signaling
OA Biochemistry
Abstract: Introduction: A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The real-time kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in vitro, and because cytokines receptors differentially utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-gamma) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-gamma receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-gamma signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Toward the Development of Real-Time Cellular Kinetic Assays to Characterize Inhibitors of Cytokine Signaling
OA Biochemistry
Abstract: Introduction: A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The real-time kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in vitro, and because cytokines receptors differentially utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-gamma) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-gamma receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-gamma signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.
Ligand-Independant Interaction of the Type I Interferon Receptor Complex is Necessary to Observe Its Biological Activity
Cytokine
Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-aR1 and IFN-aR2c, or IFN-lR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-aR1 and IFN-aR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-aR1 and IFN-aR2c were sensitive to IFN-alpha only when IFN-aR1 and IFN-aR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-aR1 and IFN-aR2c. Both IFN-aR1 and IFN-aR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Toward the Development of Real-Time Cellular Kinetic Assays to Characterize Inhibitors of Cytokine Signaling
OA Biochemistry
Abstract: Introduction: A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The real-time kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in vitro, and because cytokines receptors differentially utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-gamma) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-gamma receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-gamma signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.
Ligand-Independant Interaction of the Type I Interferon Receptor Complex is Necessary to Observe Its Biological Activity
Cytokine
Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-aR1 and IFN-aR2c, or IFN-lR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-aR1 and IFN-aR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-aR1 and IFN-aR2c were sensitive to IFN-alpha only when IFN-aR1 and IFN-aR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-aR1 and IFN-aR2c. Both IFN-aR1 and IFN-aR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.
Jak1 as the Architect for Assembling and Activating Multi-Chain Cytokine receptor Complexes in Lipid Rafts
Nova Scientific Publishers, Inc., Biochemistry Research Trends
Lipid rafts are nanometer-sized subdomains of the plasma membrane containing higher concentrations of cholesterol, phosphatidylinositols, and sphingolipids. Their lipid constituents have less conformational freedom and compact together more efficiently. Furthermore, lipids and proteins embedded within lipid rafts collide with molecular neighbors more frequently. Similar to how oil droplets form “floating islands” on water, lipid rafts form “islands” in plasma membranes. It is now clear that the integrity of these lipid rafts is essential in order to have proper signaling by many immune, neuronal, and endocrine receptor complexes and acylated proteins. The fact that these receptors co-purify with detergent-resistant membranes supports theories that they reside within lipid rafts. Because lipid rafts are too small to see directly, and change their size and position easily, they are incredibly hard to visualize, especially in intact cells. We attempted to use fluorescence resonance energy transfer to investigate the influence of the lipid raft nanoenvironment on the assembly of Class II cytokine receptor complexes, especially interferon receptors. In this chapter, we summarize our observations that (1) receptor preassembly is required for biological function, (2) the interaction between receptor chains requires both the presence of Jak1 and their co-nanolocalization within lipid rafts, (3) a sequence-supported structural analysis of Janus kinases that suggests a significant influence of phospholipids on Janus kinase function, and (4) critical observations made by others. Altogether, we present a model that lipid rafts shape the conformation of Jak1, which in turn controls how it interacts with multiple cytokine receptors, permitting their interaction and consequently their biological function. The increased viscosity of lipid rafts compared to non-raft domains may help stabilize interactions within the receptor complex.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Toward the Development of Real-Time Cellular Kinetic Assays to Characterize Inhibitors of Cytokine Signaling
OA Biochemistry
Abstract: Introduction: A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The real-time kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in vitro, and because cytokines receptors differentially utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-gamma) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-gamma receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-gamma signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.
Ligand-Independant Interaction of the Type I Interferon Receptor Complex is Necessary to Observe Its Biological Activity
Cytokine
Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-aR1 and IFN-aR2c, or IFN-lR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-aR1 and IFN-aR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-aR1 and IFN-aR2c were sensitive to IFN-alpha only when IFN-aR1 and IFN-aR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-aR1 and IFN-aR2c. Both IFN-aR1 and IFN-aR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.
Jak1 as the Architect for Assembling and Activating Multi-Chain Cytokine receptor Complexes in Lipid Rafts
Nova Scientific Publishers, Inc., Biochemistry Research Trends
Lipid rafts are nanometer-sized subdomains of the plasma membrane containing higher concentrations of cholesterol, phosphatidylinositols, and sphingolipids. Their lipid constituents have less conformational freedom and compact together more efficiently. Furthermore, lipids and proteins embedded within lipid rafts collide with molecular neighbors more frequently. Similar to how oil droplets form “floating islands” on water, lipid rafts form “islands” in plasma membranes. It is now clear that the integrity of these lipid rafts is essential in order to have proper signaling by many immune, neuronal, and endocrine receptor complexes and acylated proteins. The fact that these receptors co-purify with detergent-resistant membranes supports theories that they reside within lipid rafts. Because lipid rafts are too small to see directly, and change their size and position easily, they are incredibly hard to visualize, especially in intact cells. We attempted to use fluorescence resonance energy transfer to investigate the influence of the lipid raft nanoenvironment on the assembly of Class II cytokine receptor complexes, especially interferon receptors. In this chapter, we summarize our observations that (1) receptor preassembly is required for biological function, (2) the interaction between receptor chains requires both the presence of Jak1 and their co-nanolocalization within lipid rafts, (3) a sequence-supported structural analysis of Janus kinases that suggests a significant influence of phospholipids on Janus kinase function, and (4) critical observations made by others. Altogether, we present a model that lipid rafts shape the conformation of Jak1, which in turn controls how it interacts with multiple cytokine receptors, permitting their interaction and consequently their biological function. The increased viscosity of lipid rafts compared to non-raft domains may help stabilize interactions within the receptor complex.
Analytical Use of Multi-protein Fluorescence Resonance Energy Transfer to Demonstrate Membrane-Facilitated Interactions Within Cytokine Receptor Complexes
Cytokine
Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-gR1, IL-10R1 and IFN-lR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-lR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-gR1 and IFN-gR2 or between IFN-aR1 and IFN-aR2c increases, FRET among other pairs of proteins changes within each complex.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (1st ed.)
Nova Science Publishers, Inc.
Abstract: A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, and targeted RNA degradation. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of vector systems to diversify and optimize their utility, including naked DNA, viral vectors or genetic recombination.
Improving Analysis of Fluorescence Resonance Energy Transfer in Live Cells: Application to Fluorescent Protein-Labeled Interferon Receptors
Cytokine
The observed Fluorescence Resonance Energy Transfer (FRET) between fluorescently labeled proteins varies in cells. To understand how this variation affects our interpretation of how proteins interact in cells, we developed a protocol that mathematically separates donor-independent and donor-dependent excitations of acceptor, determines the electromagnetic interaction of donors and acceptors, and quantifies the efficiency of the interaction of donors and acceptors. By analyzing large populations of cells, we found that misbalanced or insufficient expression of acceptor or donor as well as their inefficient or reversible interaction influenced FRET efficiency in vivo. Use of red-shifted donors and acceptors gave spectra with less endogenous fluorescence but produced lower FRET efficiency, possibly caused by reduced quenching of red-shifted fluorophores in cells. Additionally, cryptic interactions between jellyfish FPs artefactually increased the apparent FRET efficiency. Our protocol can distinguish specific and nonspecific protein interactions even within highly constrained environments as plasma membranes. Overall, accurate FRET estimations in cells or within complex environments can be obtained by a combination of proper data analysis, study of sufficient numbers of cells, and use of properly empirically developed fluorescent proteins.
Is Stat1 Activated Catalytically Within Cells By the IFN-gamma Receptor Complex?
OA Biotechnology
STRUCTURED ABSTRACT: Introduction: Two distinct models of the activation of Stat1 by the interferon-gamma (IFN-gamma) receptor complex attempt to explain how activated Stat1 translocates to the nucleus to modify gene activity. Most analyses of Stat1 activation focus only on the amount of activated Stat1. Analysis of early timepoints of Stat1 activation has not been satisfactorily done, and may help refine models of Stat1 activation. The Hypothesis: If individual receptor complexes activate many Stat1 molecules over a long period of time, then the progressive activation of IFN-gamma complexes in cells (by gradual binding of IFN-gamma to its cellular receptors) will result in a parabolic initial activation of Stat1 over time. However, if individual receptor complexes activate only a limited number of Stat1 molecules or are quickly inactivated, Stat1 activation will occur linearly with time. Evaluation of Hypothesis: The initial time-resolved activation of Stat1 was investigated in response to various concentrations of IFN-gamma in certain cell lines. Parabolic Stat1 activation was seen; lower IFN-gamma concentrations correlated with longer parabolic Stat1 activation. The values of various kinetic enzymatic parameters were estimated. The latency observed in epithelial cell lines is not consistent with progressive receptor activation, but rather with a secondary superactivation. Conclusion: Individual receptor complexes activate many Stat1 molecules. Mathematical modeling suggests that (1) binding of Stat1 to the receptor complex limits the overall rate of cellular Stat1 activation, and (2) an enzymatic superactivation of ligand-bound receptor complexes can occur. This kinetic model can be used to analyze the effects of specific inhibitors on IFN-gamma signaling in mechanistic detail, or can be adapted to other cytokine signaling systems.
Cut, copy, move, delete: The study of human interferon genes reveal multiple mechanisms underlying their evolution in amniotes
Cytokine
Interferons (IFNs) are rapidly evolving cytokines released when viral infections are detected in cells. Previous research suggests that genes encoding IFNs and their receptors duplicated extensively throughout vertebrate evolution. We present molecular genetic evidence that supports the use of nonallelic homologous recombination (NAHR) to expand select IFN genes during amniote evolution. The duplication of long regions of genome (encompassing at least one functional IFN gene) followed by the insertion of this genome fragment near its parent’s location, is commonly observed in many amniote genomes. Duplicates inserted away from duplication hotspots are not as frequently perturbed with new duplicates, and tend to survive long periods of evolution, sometimes becoming new IFN subtypes. Although most duplicates are inserted parallel to and near the original sequence, the insertion of the Kelch-like 9 gene within the Type I IFN locus of placental mammals promoted antiparallel insertion of gene duplicates between the Kelch-like 9 and IFN-ε loci. Genetic exchange between highly similar Type I gene duplicates as well as between Type III IFN gene duplicates homogenized their diversification. Oddly, Type III IFN genes migrated long distances throughout the genome more frequently than did Type I IFN genes. The inter-chromosomal movement of Type I IFN genes in amniotes correlated with complete intron loss in their gene structure, and repeatedly occurred with occasional Type III IFN genes.
Development of Multifunctional Plasmids for Diverse Biotechnological Applications (2nd ed.)
Nova Science Publishers, Inc.
A bewildering variety of plasmids have been developed for a wide array of purposes in biotechnology and biomedical research. These purposes include transient and stable protein expression, mutation, recombination-mediated nuclear gene knockout, promoter analysis, RNA stability, targeted RNA degradation, and gene therapy. Each plasmid has been optimized for only one of these functions, and consequentially, convenient restriction enzyme sites surround only one genetic element. However, an optimal expression system often requires the use of different genetic elements that are rarely found on the same plasmid (such as differing promoters or eukaryotic antibiotic resistance), and exchange of these elements between plasmids is difficult. We retrofitted pcDNA3 (an archaic but well-validated and useful plasmid from Stratagene, Inc.) to facilitate the exchange of various genetic elements by inserting useful restriction endonuclease sites at the borders of these elements. With the retrofitted plasmid, called pc3.5, we can exchange not only the ectopically expressed gene of interest but also promoters or polyadenylation sites of the gene of interest, as well as the eukaryotic antibiotic resistance gene, its promoter or polyadenylation sequences. We have used derivatives of these plasmids for transient and stable gene expression, promoter analysis and induction, and mesenchymal stem cell engineering. Retrofitting plasmids is a simple process than can be applied to a wide variety of plasmid or viral vector systems to diversify and optimize their utility.
Toward the Development of Real-Time Cellular Kinetic Assays to Characterize Inhibitors of Cytokine Signaling
OA Biochemistry
Abstract: Introduction: A quantitative analysis of the activation of Stat proteins by the Janus (Jak) kinases, the enzymatic arm of cytokine receptor complexes, is finally possible due to the recent development of selective high-affinity inhibitors to each Janus kinase. The real-time kinetic analyses of Janus kinases so far utilize an in vitro system. However, because the regulation and activation of Janus kinases is more complex in cells than in vitro, and because cytokines receptors differentially utilize their Janus kinases, a kinetic analysis of signaling within cells is necessary. Results: Various types of real-time assays are discussed. To address the need for real-time analyses of Janus kinases in cells, we developed a kinetic model of the activation the Jak/Stat pathway by the interferon-gamma (IFN-gamma) receptor complex in cells. Fortunately, a high-quality real-time noninvasive cellular assay system is possible because Stat proteins are catalytically activated by the IFN-gamma receptor complexes. Discussion: With a real time cellular assay, we can use selective Jak inhibitors to identify how and when kinases participate in the various stages of IFN-gamma signaling, as well as to establish how much of a given inhibitor may be necessary to inhibit or modify signaling by particular cytokines. We can also investigate the time-resolved mechanism of action of new receptor-specific antagonists.
Ligand-Independant Interaction of the Type I Interferon Receptor Complex is Necessary to Observe Its Biological Activity
Cytokine
Ectopic coexpression of the two chains of the Type I and Type III interferon (IFN) receptor complexes (IFN-aR1 and IFN-aR2c, or IFN-lR1 and IL-10R2) yielded sensitivity to IFN-alpha or IFN-lambda in only some cells. We found that IFN-aR1 and IFN-aR2c exhibit FRET only when expressed at equivalent and low levels. Expanded clonal cell lines expressing both IFN-aR1 and IFN-aR2c were sensitive to IFN-alpha only when IFN-aR1 and IFN-aR2c exhibited FRET in the absence of human IFN-alpha. Coexpression of RACK-1 or Jak1 enhanced the affinity of the interaction between IFN-aR1 and IFN-aR2c. Both IFN-aR1 and IFN-aR2c exhibited FRET with Jak1 and Tyk2. Together with data showing that disruption of the preassociation between the IFN-gamma receptor chains inhibited its biological activity, we propose that biologically active IFN receptors require ligand-independent juxtaposition of IFN receptor chains assisted by their associated cytosolic proteins.
Jak1 as the Architect for Assembling and Activating Multi-Chain Cytokine receptor Complexes in Lipid Rafts
Nova Scientific Publishers, Inc., Biochemistry Research Trends
Lipid rafts are nanometer-sized subdomains of the plasma membrane containing higher concentrations of cholesterol, phosphatidylinositols, and sphingolipids. Their lipid constituents have less conformational freedom and compact together more efficiently. Furthermore, lipids and proteins embedded within lipid rafts collide with molecular neighbors more frequently. Similar to how oil droplets form “floating islands” on water, lipid rafts form “islands” in plasma membranes. It is now clear that the integrity of these lipid rafts is essential in order to have proper signaling by many immune, neuronal, and endocrine receptor complexes and acylated proteins. The fact that these receptors co-purify with detergent-resistant membranes supports theories that they reside within lipid rafts. Because lipid rafts are too small to see directly, and change their size and position easily, they are incredibly hard to visualize, especially in intact cells. We attempted to use fluorescence resonance energy transfer to investigate the influence of the lipid raft nanoenvironment on the assembly of Class II cytokine receptor complexes, especially interferon receptors. In this chapter, we summarize our observations that (1) receptor preassembly is required for biological function, (2) the interaction between receptor chains requires both the presence of Jak1 and their co-nanolocalization within lipid rafts, (3) a sequence-supported structural analysis of Janus kinases that suggests a significant influence of phospholipids on Janus kinase function, and (4) critical observations made by others. Altogether, we present a model that lipid rafts shape the conformation of Jak1, which in turn controls how it interacts with multiple cytokine receptors, permitting their interaction and consequently their biological function. The increased viscosity of lipid rafts compared to non-raft domains may help stabilize interactions within the receptor complex.
Analytical Use of Multi-protein Fluorescence Resonance Energy Transfer to Demonstrate Membrane-Facilitated Interactions Within Cytokine Receptor Complexes
Cytokine
Experiments measuring Fluorescence Resonance Energy Transfer (FRET) between cytokine receptor chains and their associated proteins led to hypotheses describing their organization in intact cells. These interactions occur within a larger protein complex or within a given nano-environment. To illustrate this complexity empirically, we developed a protocol to analyze FRET among more than two fluorescent proteins (multi-FRET). In multi-FRET, we model FRET among more than two fluorophores as the sum of all possible pairwise interactions within the complex. We validated our assumption by demonstrating that FRET among pairs within a fluorescent triplet resembled FRET between each pair measured in the absence of the third fluorophore. FRET between two receptor chains increases with increasing FRET between the ligand-binding chain (e.g., IFN-gR1, IL-10R1 and IFN-lR1) and an acylated fluorescent protein that preferentially resides within subsections of the plasma membrane. The interaction of IL-10R2 with IFN-lR1 or IL-10R1 results in decreased FRET between IL-10R2 and the acylated fluorescent protein. Finally, we analyzed FRET among four fluorescent proteins to demonstrate that as FRET between IFN-gR1 and IFN-gR2 or between IFN-aR1 and IFN-aR2c increases, FRET among other pairs of proteins changes within each complex.
Intron loss in interferon genes follows a distinct set of stages, and may confer an evolutionary advantage
Cytokine
The promoter-intron-exon structure of genes evolve. While the structures of some IFN genes (e.g., piscine and amphibian Type I IFNs, most tetrapod IFN-λ genes) resemble those of other class II cytokines (e.g., interleukins-10, 19, 20, 22, 24, 26), the structures of other IFN genes differ significantly. Although all bony vertebrate IFN-γ genes lack the canonical third intron, and all amniote Type I IFN genes lack introns, only some IFN-λ genes lost their introns. Interestingly, these intronless IFN-λ genes are not preferentially related to one another nor are they clustered with canonical multi-intron IFN-λ genes. Hypothesizing that intronless IFN-λ genes repeatedly and independently evolved and transposed throughout the genome, we sought to understand the genetic processes involved in their intron loss and genomic migration. Utilizing the high conservation of the promoters, the UTRs and the ORFs of the IFN-λ genes, we collected data from two families of intronless IFN-λ genes, and developed a model supported by these data to explain how intronless IFN-λ genes evolved. (1) A cytoplasmic IFN-λ cDNA generated by reverse transcriptional activity enters the nucleus and attempts to recombine with its multi-exon progenitor. (2) Nuclear DNA synthesis at the 5' and 3' ends within recombination intermediates affixes the promoter onto the cDNA and preserves its 3' UTR. (3) Resolution of the recombination complex releases the promoter-associated cDNA. (4) The released intronless gene co-integrates with a highly duplicated sequence undergoing transposition. We propose that this process explains not only the evolution of the gene structure of IFN genes, but also the increased transposition of intronless genes in genomes, and may confer an evolutionary advantage.
