Journal Articles
Crosstalk between Hippo and TGFβ: Subcellular Localization of YAP/TAZ/Smad Complexes
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Karin Grannas, Linda Arngården, Peter Lönn, Magdalena Mazurkiewicz, Andries Blokzijl, Agata Zieba, Ola Söderberg
The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ induces formation of YAP/TAZ–Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ–Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP–Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ–Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFβ signaling pathways.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Karin Grannas, Linda Arngården, Peter Lönn, Magdalena Mazurkiewicz, Andries Blokzijl, Agata Zieba, Ola Söderberg
The Hippo pathway plays a crucial role in growth control, proliferation and tumor suppression. Activity of the signaling pathway is associated with cell density sensing and tissue organization. Furthermore, the Hippo pathway helps to coordinate cellular processes through crosstalk with growth-factor-mediated signaling pathways such as TGFβ. Here we have examined the localization of interactions between proteins of the Hippo pathway (YAP/TAZ) and TGFβ (Smad2/3) signaling pathway by using in situ proximity ligation assays. We investigated the formation of protein complexes between YAP/TAZ and Smad2/3 and examined how these interactions were affected by TGFβ stimulation and cell density in HaCaT keratinocytes and in Smad4-deficient HT29 colon cancer cells. We demonstrate that TGFβ induces formation of YAP/TAZ–Smad2/3 complexes in HaCaT cells. Under sparse cell conditions, the complexes were detected to a higher degree and were predominantly located in the nucleus, while under dense culture conditions, the complexes were fewer and mainly located in the cytoplasm. Surprisingly, we could not detect any YAP/TAZ–Smad2/3 complexes in HT29 cells. To examine if Smad4 deficiency was responsible for the absence of interactions, we treated HaCaT cells with siRNA targeting Smad4. However, we could still observe complex formation in the siRNA-treated cells, suggesting that Smad4 is not essential for the YAP–Smad2/3 interaction. In conclusion, this study shows localized, density-dependent formation of YAP/TAZ–Smad2/3 complexes in HaCaT cells and provides evidence supporting a crosstalk between the Hippo and the TGFβ signaling pathways.
Graphical abstract
Categories: Journal Articles
Studying Cellular Signal Transduction with OMIC Technologies
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Benjamin D. Landry, David C. Clarke, Michael J. Lee
In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly non-linear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and “OMIC” approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the “signal” can take different forms in different situations. Signals are encoded in diverse ways such as protein–protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases, signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the “right” experiments and the “right” modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Benjamin D. Landry, David C. Clarke, Michael J. Lee
In the gulf between genotype and phenotype exists proteins and, in particular, protein signal transduction systems. These systems use a relatively limited parts list to respond to a much longer list of extracellular, environmental, and/or mechanical cues with rapidity and specificity. Most signaling networks function in a highly non-linear and often contextual manner. Furthermore, these processes occur dynamically across space and time. Because of these complexities, systems and “OMIC” approaches are essential for the study of signal transduction. One challenge in using OMIC-scale approaches to study signaling is that the “signal” can take different forms in different situations. Signals are encoded in diverse ways such as protein–protein interactions, enzyme activities, localizations, or post-translational modifications to proteins. Furthermore, in some cases, signals may be encoded only in the dynamics, duration, or rates of change of these features. Accordingly, systems-level analyses of signaling may need to integrate multiple experimental and/or computational approaches. As the field has progressed, the non-triviality of integrating experimental and computational analyses has become apparent. Successful use of OMIC methods to study signaling will require the “right” experiments and the “right” modeling approaches, and it is critical to consider both in the design phase of the project. In this review, we discuss common OMIC and modeling approaches for studying signaling, emphasizing the philosophical and practical considerations for effectively merging these two types of approaches to maximize the probability of obtaining reliable and novel insights into signaling biology.
Graphical abstract
Categories: Journal Articles
Integrating -Omics: Systems Biology as Explored Through C. elegans Research
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Roel Van Assche, Valérie Broeckx, Kurt Boonen, Evelyne Maes, Wouter De Haes, Liliane Schoofs, Liesbet Temmerman
-Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration rather than simply interpreting single -ome studies. This review evaluates the application of integrated -omics approaches with a focus on Caenorhabditis elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 21
Author(s): Roel Van Assche, Valérie Broeckx, Kurt Boonen, Evelyne Maes, Wouter De Haes, Liliane Schoofs, Liesbet Temmerman
-Omics data have become indispensable to systems biology, which aims to describe the full complexity of functional cells, tissues, organs and organisms. Generating vast amounts of data via such methods, researchers have invested in ways of handling and interpreting these. From the large volumes of -omics data that have been gathered over the years, it is clear that the information derived from one -ome is usually far from complete. Now, individual techniques and methods for integration are maturing to the point that researchers can focus on network-based integration rather than simply interpreting single -ome studies. This review evaluates the application of integrated -omics approaches with a focus on Caenorhabditis elegans studies, intending to direct researchers in this field to useful databases and inspiring examples.
Graphical abstract
Categories: Journal Articles
Special Issue OMICS Approaches to Unravel Molecular Function
Publication date: 23 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 21
Source:Journal of Molecular Biology, Volume 427, Issue 21
Categories: Journal Articles
Origins of Programmable Nucleases for Genome Engineering
Publication date: Available online 23 October 2015
Source:Journal of Molecular Biology
Author(s): Srinivasan Chandrasegaran, Dana Carroll
Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) that showed that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins (ZFPs) and FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18 bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination (HR) in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain, expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system - the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein - have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues like efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk vs. benefit analysis and informed consent.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Srinivasan Chandrasegaran, Dana Carroll
Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) that showed that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins (ZFPs) and FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18 bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination (HR) in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain, expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system - the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein - have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues like efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk vs. benefit analysis and informed consent.
Graphical abstract
Categories: Journal Articles
Hsp70 and Hsp90 of E. coli Directly Interact for Collaboration in Protein Remodeling
Publication date: Available online 23 October 2015
Source:Journal of Molecular Biology
Author(s): Olivier Genest, Joel R. Hoskins, Andrea N. Kravats, Shannon M. Doyle, Sue Wickner
Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperones. We explored the collaboration between Escherichia coli Hsp90 and DnaK and found that the two chaperones form a complex that is stabilized by client protein binding. A J-domain protein, CbpA, facilitates assembly of the Hsp90Ec–DnaK–client complex. We identified E. coli Hsp90 mutants defective in DnaK interaction in vivo and show that the purified mutant proteins are defective in physical and functional interaction with DnaK. Understanding how Hsp90 and Hsp70 collaborate in protein remodeling will provide the groundwork for the development of new therapeutic strategies targeting multiple chaperones and cochaperones.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Olivier Genest, Joel R. Hoskins, Andrea N. Kravats, Shannon M. Doyle, Sue Wickner
Hsp90 is a highly conserved molecular chaperone that remodels hundreds of client proteins, many involved in the progression of cancer and other diseases. It functions with the Hsp70 chaperone and numerous cochaperones. The bacterial Hsp90 functions with an Hsp70 chaperone, DnaK, but is independent of Hsp90 cochaperones. We explored the collaboration between Escherichia coli Hsp90 and DnaK and found that the two chaperones form a complex that is stabilized by client protein binding. A J-domain protein, CbpA, facilitates assembly of the Hsp90Ec–DnaK–client complex. We identified E. coli Hsp90 mutants defective in DnaK interaction in vivo and show that the purified mutant proteins are defective in physical and functional interaction with DnaK. Understanding how Hsp90 and Hsp70 collaborate in protein remodeling will provide the groundwork for the development of new therapeutic strategies targeting multiple chaperones and cochaperones.
Graphical abstract
Categories: Journal Articles
Multiple weak linear motifs enhance recruitment and processivity in SPOP-mediated substrate ubiquitination
Publication date: Available online 22 October 2015
Source:Journal of Molecular Biology
Author(s): Wendy K. Pierce, Christy R. Grace, Jihun Lee, Amanda Nourse, Melissa R. Marzahn, Edmond R. Watson, Anthony A. High, Junmin Peng, Brenda A. Schulman, Tanja Mittag
Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert stronger physiological relevance than suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli31-90) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3SPOP and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli31-90, the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli31-90 ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers in posttranslational modification.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Wendy K. Pierce, Christy R. Grace, Jihun Lee, Amanda Nourse, Melissa R. Marzahn, Edmond R. Watson, Anthony A. High, Junmin Peng, Brenda A. Schulman, Tanja Mittag
Primary sequence motifs, with millimolar affinities for binding partners, are abundant in disordered protein regions. In multivalent interactions, such weak linear motifs can cooperate to recruit binding partners via avidity effects. If linear motifs recruit modifying enzymes, optimal placement of weak motifs may regulate access to modification sites. Weak motifs may thus exert stronger physiological relevance than suggested by their affinities, but molecular mechanisms of their function are still poorly understood. Herein, we use the N-terminal disordered region of the Hedgehog transcriptional regulator Gli3 (Gli31-90) to determine the role of weak motifs encoded in its primary sequence for the recruitment of its ubiquitin ligase CRL3SPOP and the subsequent effect on ubiquitination efficiency. The substrate adaptor SPOP binds linear motifs through its MATH domain and forms higher-order oligomers through its oligomerization domains, rendering SPOP multivalent for its substrates. Gli3 has multiple weak SPOP binding motifs. We map three such motifs in Gli31-90, the weakest of which has a millimolar dissociation constant. Multivalency of ligase and substrate for each other facilitates enhanced ligase recruitment and stimulates Gli31-90 ubiquitination in in vitro ubiquitination assays. We speculate that the weak motifs enable processivity through avidity effects and by providing steric access to lysine residues that are otherwise not prioritized for polyubiquitination. Weak motifs may generally be employed in multivalent systems to act as gatekeepers in posttranslational modification.
Graphical abstract
Categories: Journal Articles
Crystal Structure of the Human tRNA m1A58 Methyltransferase–tRNA3Lys Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target
Publication date: Available online 22 October 2015
Source:Journal of Molecular Biology
Author(s): Janet Finer-Moore, Nadine Czudnochowski, Joseph D. O'Connell, Amy Liya Wang, Robert M. Stroud
Human tRNA3 Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3 Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-l-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3 Lys and the product S-adenosyl-l-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Janet Finer-Moore, Nadine Czudnochowski, Joseph D. O'Connell, Amy Liya Wang, Robert M. Stroud
Human tRNA3 Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3 Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-l-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3 Lys and the product S-adenosyl-l-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.
Graphical abstract
Categories: Journal Articles
Predicting Large RNA-Like Topologies by a Knowledge-Based Clustering Approach
Publication date: Available online 22 October 2015
Source:Journal of Molecular Biology
Author(s): Naoto Baba, Shereef Elmetwaly, Namhee Kim, Tamar Schlick
An analysis and expansion of our resource for classifying, predicting, and designing RNA structures, RAG (RNA-As-Graphs), is presented, with the goal of understanding features of RNA-like and non-RNA-like motifs and exploiting this information for RNA design. RAG was first reported in 2004 for cataloguing RNA secondary structure motifs using graph representations. In 2011, the RAG resource was updated with the increased availability of RNA structures and improved by utilities for analyzing RNA structures, including substructuring and search tools. We also classified RNA structures as graphs up to 10 vertices (~200 nucleotides) as three classes: existing, RNA-like, and non-RNA-like using clustering approaches. Here, we focus on the tree graphs and evaluate the newly founded RNAs since 2011, which also support our refined predictions of RNA-like motifs. We expand the RAG resource for large tree graphs up to 13 vertices (~260 nucleotides), thereby cataloguing more than 10 times as many secondary structures. We apply clustering algorithms based on features of RNA secondary structures translated from known tertiary structures to suggest which large RNA motifs can be considered “RNA-like”. The results by the Partitioning Around Medoids (PAM) approach, in particular, reveal good accuracy, with small error for the largest cases. The RAG update here up to 13 vertices offers a useful graph-based tool for exploring RNA motifs and suggesting large RNA motifs for design.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Naoto Baba, Shereef Elmetwaly, Namhee Kim, Tamar Schlick
An analysis and expansion of our resource for classifying, predicting, and designing RNA structures, RAG (RNA-As-Graphs), is presented, with the goal of understanding features of RNA-like and non-RNA-like motifs and exploiting this information for RNA design. RAG was first reported in 2004 for cataloguing RNA secondary structure motifs using graph representations. In 2011, the RAG resource was updated with the increased availability of RNA structures and improved by utilities for analyzing RNA structures, including substructuring and search tools. We also classified RNA structures as graphs up to 10 vertices (~200 nucleotides) as three classes: existing, RNA-like, and non-RNA-like using clustering approaches. Here, we focus on the tree graphs and evaluate the newly founded RNAs since 2011, which also support our refined predictions of RNA-like motifs. We expand the RAG resource for large tree graphs up to 13 vertices (~260 nucleotides), thereby cataloguing more than 10 times as many secondary structures. We apply clustering algorithms based on features of RNA secondary structures translated from known tertiary structures to suggest which large RNA motifs can be considered “RNA-like”. The results by the Partitioning Around Medoids (PAM) approach, in particular, reveal good accuracy, with small error for the largest cases. The RAG update here up to 13 vertices offers a useful graph-based tool for exploring RNA motifs and suggesting large RNA motifs for design.
Graphical abstract
Categories: Journal Articles
HSFs, Stress Sensors and Sculptors of Transcription Compartments and Epigenetic Landscapes
Publication date: Available online 22 October 2015
Source:Journal of Molecular Biology
Author(s): Federico Miozzo, Délara Sabéran-Djoneidi, Valérie Mezger
Starting as a paradigm for stress responses, the study of the transcription factor (TF) family of heat shock factors (HSFs) has quickly and widely expanded these last decades, thanks to their fascinating and significant involvement in a variety of pathophysiological processes, including development, reproduction, neurodegeneration and carcinogenesis. HSFs, originally defined as classical TFs, strikingly appeared to play a central and often pioneering role in reshaping the epigenetic landscape. In this review, we describe how HSFs are able to sense the epigenetic environment, and we review recent data that support their role as sculptors of the chromatin landscape through their complex interplay with chromatin remodelers, histone-modifying enzymes and non-coding RNAs.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Federico Miozzo, Délara Sabéran-Djoneidi, Valérie Mezger
Starting as a paradigm for stress responses, the study of the transcription factor (TF) family of heat shock factors (HSFs) has quickly and widely expanded these last decades, thanks to their fascinating and significant involvement in a variety of pathophysiological processes, including development, reproduction, neurodegeneration and carcinogenesis. HSFs, originally defined as classical TFs, strikingly appeared to play a central and often pioneering role in reshaping the epigenetic landscape. In this review, we describe how HSFs are able to sense the epigenetic environment, and we review recent data that support their role as sculptors of the chromatin landscape through their complex interplay with chromatin remodelers, histone-modifying enzymes and non-coding RNAs.
Graphical abstract
Categories: Journal Articles
Structure of Zeste–DNA Complex Reveals a New Modality of DNA Recognition by Homeodomain-Like Proteins
Publication date: Available online 21 October 2015
Source:Journal of Molecular Biology
Author(s): Guan-Nan Gao, Mingzhu Wang, Na Yang, Ying Huang, Rui-Ming Xu
Drosophila Zeste is a DNA binding protein important for chromatin-targeted regulation of gene expression. It is best studied in the context of transvection—a mechanism of interallelic gene regulation involving paired chromosomes—and repression of the expression of white by Zeste mutants. Both of these functions depend on the DNA binding and self-association properties of Zeste, but the underlying structural basis remains unknown. Here we report the crystal structure of the DNA binding domain of Zeste in complex with a 19-bp DNA duplex containing the consensus recognition sequence motif. The structure reveals a helix–turn–helix Myb/homeodomain-like fold with the Zeste-specific insertion sequence forming a short helix and a long loop. Direct base contacts by the major groove binding helix principally account for the sequence-specific recognition, and backbone contacts via the Zeste-specific insertion are mainly responsible for the length requirement and the orientation of DNA. Our structural and biochemical characterizations of the DNA binding property of Zeste uncover an altered DNA binding modality of homeodomain-like proteins, and the structural information should facilitate the unraveling of the intricate mechanism of Zeste in regulation of gene expression.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Guan-Nan Gao, Mingzhu Wang, Na Yang, Ying Huang, Rui-Ming Xu
Drosophila Zeste is a DNA binding protein important for chromatin-targeted regulation of gene expression. It is best studied in the context of transvection—a mechanism of interallelic gene regulation involving paired chromosomes—and repression of the expression of white by Zeste mutants. Both of these functions depend on the DNA binding and self-association properties of Zeste, but the underlying structural basis remains unknown. Here we report the crystal structure of the DNA binding domain of Zeste in complex with a 19-bp DNA duplex containing the consensus recognition sequence motif. The structure reveals a helix–turn–helix Myb/homeodomain-like fold with the Zeste-specific insertion sequence forming a short helix and a long loop. Direct base contacts by the major groove binding helix principally account for the sequence-specific recognition, and backbone contacts via the Zeste-specific insertion are mainly responsible for the length requirement and the orientation of DNA. Our structural and biochemical characterizations of the DNA binding property of Zeste uncover an altered DNA binding modality of homeodomain-like proteins, and the structural information should facilitate the unraveling of the intricate mechanism of Zeste in regulation of gene expression.
Graphical abstract
Categories: Journal Articles
Insights into eukaryotic translation initiation from mass spectrometry of macromolecular protein assemblies
Publication date: Available online 21 October 2015
Source:Journal of Molecular Biology
Author(s): Carla Schmidt, Victoria Beilsten-Edmands, Carol V. Robinson
Translation initiation in eukaryotes requires the interplay of at least ten initiation factors which interact at the different steps of this phase of gene expression. The interactions of initiation factors and related proteins are in general controlled by phosphorylation, which serves as a regulatory switch to turn protein translation on or off. To fully understand these processes, the structures of initiation factors and a complete description of their post-translational modifications status is therefore required. In recent years, mass spectrometry has contributed considerably to provide this information and nowadays is proving to be indispensable when studying dynamic heterogeneous protein complexes such as the eukaryotic initiation factors. Herein, we highlight mass spectrometric approaches commonly applied to identify interacting subunits and their post-translational modifications and the structural techniques which allow the architecture of protein complexes to be assessed. We present recent structural investigations of initiation factors, their interactions with other factors and with ribosomes and assess the models generated. These models allow us to locate post-translational modifications within initiation factor complexes and to highlight possible roles for phosphorylation sites in regulating interaction interfaces.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Carla Schmidt, Victoria Beilsten-Edmands, Carol V. Robinson
Translation initiation in eukaryotes requires the interplay of at least ten initiation factors which interact at the different steps of this phase of gene expression. The interactions of initiation factors and related proteins are in general controlled by phosphorylation, which serves as a regulatory switch to turn protein translation on or off. To fully understand these processes, the structures of initiation factors and a complete description of their post-translational modifications status is therefore required. In recent years, mass spectrometry has contributed considerably to provide this information and nowadays is proving to be indispensable when studying dynamic heterogeneous protein complexes such as the eukaryotic initiation factors. Herein, we highlight mass spectrometric approaches commonly applied to identify interacting subunits and their post-translational modifications and the structural techniques which allow the architecture of protein complexes to be assessed. We present recent structural investigations of initiation factors, their interactions with other factors and with ribosomes and assess the models generated. These models allow us to locate post-translational modifications within initiation factor complexes and to highlight possible roles for phosphorylation sites in regulating interaction interfaces.
Graphical abstract
Categories: Journal Articles
Pathogen Resistance Mediated by IL-22 Signaling at the Epithelial–Microbiota Interface
Publication date: Available online 21 October 2015
Source:Journal of Molecular Biology
Author(s): Fernanda Schreiber, Julia Maryam Arasteh, Trevor D. Lawley
Intestinal colonization resistance to bacterial pathogens is generally associated, among other factors, with mucosal homeostasis that preserves the integrity of the intestinal barrier. Mucosal homeostasis depends on physical and molecular interactions between three components: the resident microbiota, the epithelial layer and the local immune system. The cytokine IL-22 helps to orchestrate this three-way interaction. IL-22 is produced by immune cells present beneath the epithelium and is induced by bacteria present in the intestine. IL-22 stimulates the epithelial cells via the IL-22RA1–IL-10R2 receptor complex inducing changes in the expression of genes involved in the maintenance of epithelial barrier integrity, with a variety of functions in pathogen resistance such as mucus layer modifications and hydration, tight junction fortification and the production of a broad range of bactericidal compounds. These mechanisms of pathogen resistance, in turn, affect the microbiota composition and create an environment that excludes pathogens. Here we highlight the role of IL-22 as key mediator in the give-and-take relationship between the microbiota and the host that impacts pathogen resistance.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Fernanda Schreiber, Julia Maryam Arasteh, Trevor D. Lawley
Intestinal colonization resistance to bacterial pathogens is generally associated, among other factors, with mucosal homeostasis that preserves the integrity of the intestinal barrier. Mucosal homeostasis depends on physical and molecular interactions between three components: the resident microbiota, the epithelial layer and the local immune system. The cytokine IL-22 helps to orchestrate this three-way interaction. IL-22 is produced by immune cells present beneath the epithelium and is induced by bacteria present in the intestine. IL-22 stimulates the epithelial cells via the IL-22RA1–IL-10R2 receptor complex inducing changes in the expression of genes involved in the maintenance of epithelial barrier integrity, with a variety of functions in pathogen resistance such as mucus layer modifications and hydration, tight junction fortification and the production of a broad range of bactericidal compounds. These mechanisms of pathogen resistance, in turn, affect the microbiota composition and create an environment that excludes pathogens. Here we highlight the role of IL-22 as key mediator in the give-and-take relationship between the microbiota and the host that impacts pathogen resistance.
Graphical abstract
Categories: Journal Articles
Structure of Full-Length Human PDGFRβ Bound to Its Activating Ligand PDGF-B as Determined by Negative-Stain Electron Microscopy
Publication date: Available online 20 October 2015
Source:Journal of Molecular Biology
Author(s): Po-Han Chen, Vinzenz Unger, Xiaolin He
Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the unregulated states, cancer formation. One long-standing puzzle regarding RTKs centers on how the extracellular domain (ECD), which detects and binds to growth factors, is coupled with the intracellular domain kinase activation. While extensive structural works on the soluble portions of RTKs have provided critical insights into RTK structures and functions, lack of a full-length receptor structure has hindered a comprehensive overview of RTK activation. In this study, we successfully purified and determined a 27-Å-resolution structure of PDGFRβ [a full-length human platelet-derived growth factor receptor], in complex with its ligand PDGF-B. In the ligand-stimulated complex, two PDGFRβs assemble into a dimer via an extensive interface essentially running along the full-length of the receptor, suggesting that the membrane-proximal region, the transmembrane helix and the kinase domain of PDGFRβ are involved in dimerization. Major structural differences are seen between the full-length and soluble ECD structures, rationalizing previous experimental data on how membrane-proximal domains modulate receptor ligand-binding affinity and dimerization efficiency. Also, in contrast to the 2-fold symmetry of the ECD, the intracellular kinase domains adopt an asymmetric dimer arrangement, in agreement with prior observations for the closely related KIT receptor. In essence, the structure provides a first glimpse into how platelet-derived growth factor receptor ECD, upon ligand stimulation, is coupled to its intracellular domain kinase activation.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Po-Han Chen, Vinzenz Unger, Xiaolin He
Members of the receptor tyrosine kinases (RTKs) regulate important cellular functions such as cell growth and migration, which are key steps in angiogenesis, in organ morphogenesis and in the unregulated states, cancer formation. One long-standing puzzle regarding RTKs centers on how the extracellular domain (ECD), which detects and binds to growth factors, is coupled with the intracellular domain kinase activation. While extensive structural works on the soluble portions of RTKs have provided critical insights into RTK structures and functions, lack of a full-length receptor structure has hindered a comprehensive overview of RTK activation. In this study, we successfully purified and determined a 27-Å-resolution structure of PDGFRβ [a full-length human platelet-derived growth factor receptor], in complex with its ligand PDGF-B. In the ligand-stimulated complex, two PDGFRβs assemble into a dimer via an extensive interface essentially running along the full-length of the receptor, suggesting that the membrane-proximal region, the transmembrane helix and the kinase domain of PDGFRβ are involved in dimerization. Major structural differences are seen between the full-length and soluble ECD structures, rationalizing previous experimental data on how membrane-proximal domains modulate receptor ligand-binding affinity and dimerization efficiency. Also, in contrast to the 2-fold symmetry of the ECD, the intracellular kinase domains adopt an asymmetric dimer arrangement, in agreement with prior observations for the closely related KIT receptor. In essence, the structure provides a first glimpse into how platelet-derived growth factor receptor ECD, upon ligand stimulation, is coupled to its intracellular domain kinase activation.
Graphical abstract
Categories: Journal Articles
The structural basis of actin organization by vinculin and metavinculin
Publication date: Available online 20 October 2015
Source:Journal of Molecular Biology
Author(s): Laura Y. Kim, Peter M. Thompson, Hyunna T. Lee, Mihir Pershad, Sharon L. Campbell, Gregory M. Alushin
Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5Å resolution cryo-EM reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal “strap” and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1’ helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform which suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner and provide a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Laura Y. Kim, Peter M. Thompson, Hyunna T. Lee, Mihir Pershad, Sharon L. Campbell, Gregory M. Alushin
Vinculin is an essential adhesion protein that links membrane-bound integrin and cadherin receptors through their intracellular binding partners to filamentous actin, facilitating mechanotransduction. Here we present an 8.5Å resolution cryo-EM reconstruction and pseudo-atomic model of the vinculin tail (Vt) domain bound to F-actin. Upon actin engagement, the N-terminal “strap” and helix 1 are displaced from the Vt helical bundle to mediate actin bundling. We find that an analogous conformational change also occurs in the H1’ helix of the tail domain of metavinculin (MVt) upon actin binding, a muscle-specific splice isoform which suppresses actin bundling by Vt. These data support a model in which metavinculin tunes the actin bundling activity of vinculin in a tissue-specific manner and provide a mechanistic framework for understanding metavinculin mutations associated with hereditary cardiomyopathies.
Graphical abstract
Categories: Journal Articles
Tools and Principles for Microbial Gene Circuit Engineering
Publication date: Available online 20 October 2015
Source:Journal of Molecular Biology
Author(s): Robert W. Bradley, Martin Buck, Baojun Wang
Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics.
Graphical abstract
Source:Journal of Molecular Biology
Author(s): Robert W. Bradley, Martin Buck, Baojun Wang
Synthetic biologists aim to construct novel genetic circuits with useful applications through rational design and forward engineering. Given the complexity of signal processing that occurs in natural biological systems, engineered microbes have the potential to perform a wide range of desirable tasks that require sophisticated computation and control. Realising this goal will require accurate predictive design of complex synthetic gene circuits and accompanying large sets of quality modular and orthogonal genetic parts. Here we present a current overview of the versatile components and tools available for engineering gene circuits in microbes, including recently developed RNA-based tools that possess large dynamic ranges and can be easily programmed. We introduce design principles that enable robust and scalable circuit performance such as insulating a gene circuit against unwanted interactions with its context, and we describe efficient strategies for rapidly identifying and correcting causes of failure and fine-tuning circuit characteristics.
Graphical abstract
Categories: Journal Articles
Editorial Board
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20
Source:Journal of Molecular Biology, Volume 427, Issue 20
Categories: Journal Articles
Contents List
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20
Source:Journal of Molecular Biology, Volume 427, Issue 20
Categories: Journal Articles
Influence of Internal DNA Pressure on Stability and Infectivity of Phage λ
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20
Author(s): D.W. Bauer, A. Evilevitch
Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65–75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40–55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 20
Author(s): D.W. Bauer, A. Evilevitch
Viruses must remain infectious while in harsh extracellular environments. An important aspect of viral particle stability for double-stranded DNA viruses is the energetically unfavorable state of the tightly confined DNA chain within the virus capsid creating pressures of tens of atmospheres. Here, we study the influence of internal genome pressure on the thermal stability of viral particles. Using differential scanning calorimetry to monitor genome loss upon heating, we find that internal pressure destabilizes the virion, resulting in a smaller activation energy barrier to trigger DNA release. These experiments are complemented by plaque assay and electron microscopy measurements to determine the influence of intra-capsid DNA pressure on the rates of viral infectivity loss. At higher temperatures (65–75°C), failure to retain the packaged genome is the dominant mechanism of viral inactivation. Conversely, at lower temperatures (40–55°C), a separate inactivation mechanism dominates, which results in non-infectious particles that still retain their packaged DNA. Most significantly, both mechanisms of infectivity loss are directly influenced by internal DNA pressure, with higher pressure resulting in a more rapid rate of inactivation at all temperatures.
Graphical abstract
Categories: Journal Articles
Epigenetic Modulation of Human Podocyte Vitamin D Receptor in HIV Milieu
Publication date: 9 October 2015
Source:Journal of Molecular Biology, Volume 427, Issue 20
Author(s): Nirupama Chandel, Kameshwar S. Ayasolla, Xiqian Lan, Maria Sultana-Syed, Amrita Chawla, Rivka Lederman, Vasupradha Vethantham, Moin A. Saleem, Praveen N. Chander, Ashwani Malhotra, Pravin C. Singhal
HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression.
Graphical abstract
Source:Journal of Molecular Biology, Volume 427, Issue 20
Author(s): Nirupama Chandel, Kameshwar S. Ayasolla, Xiqian Lan, Maria Sultana-Syed, Amrita Chawla, Rivka Lederman, Vasupradha Vethantham, Moin A. Saleem, Praveen N. Chander, Ashwani Malhotra, Pravin C. Singhal
HIV (human immunodeficiency virus) has been reported to induce podocyte injury through down regulation of vitamin D receptor (VDR) and activation of renin angiotensin system; however, the involved mechanism is not clear. Since HIV has been reported to modulate gene expression via epigenetic phenomena, we asked whether epigenetic factors contribute to down regulation of VDR. Kidney cells in HIV transgenic mice and HIV-infected podocytes (HIV/HPs) displayed enhanced expression of SNAIL, a repressor of VDR. To elucidate the mechanism, we studied the effect of HIV on expression of molecules involved in SNAIL repressor complex formation and demonstrated that HIV enhances expression of the histone deacetylase HDAC1 and DNA methyl transferases DNMT3b and DNMT1. 293T cells, when stably transfected with SNAIL (SNAIL/293T), displayed suppressed transcription and translation of VDR. In SNAIL/293T cells, co-immunoprecipitation studies revealed the association of HDAC1, DNMT3b, DNMT1, and mSin3A with SNAIL. Chromatin immunoprecipitation experiments confirmed the presence of the SNAIL repressor complex at the VDR promoter. Consistent with the enhanced DNA methyl transferase expression in HIV/HPs, there was an increased CpG methylation at the VDR promoter. Chromatin immunoprecipitation assay confirmed occurrence of H3K4 trimethylation on SNAIL promoter. Neither a VDR agonist (VDA) nor an HDAC inhibitor (HDACI) nor a demethylating agent (DAC) individually could optimally up regulate VDR in HIV milieu. However, VDA and HDACI when combined were successful in de-repressing VDR expression. Our findings demonstrate that SNAIL recruits multiple chromatin enzymes to form a repressor complex in HIV milieu that down regulates VDR expression.
Graphical abstract
Categories: Journal Articles
