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Mass spectrometry methods to study protein-metabolite interactions.

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Mass spectrometry methods to study protein-metabolite interactions.

Expert Opin Drug Discov. 2017 Sep 21;:1-10

Authors: Guo H, Peng H, Emili A

Abstract
INTRODUCTION: To ​​​​understand and manipulate biochemical processes and signaling pathways, the knowledge of endogenous protein-metabolite interactions would be extremely helpful. Recent developments in precision mass spectrometry, high-throughput proteomics and sensitive metabolomic profiling are beginning to converge on a possible solution, heralding a new era of global metabolome-proteome 'interactome' studies that promise to change biomedical research and drug discovery. Areas covered: Here, we review innovative mass spectrometry-based methods and recent pioneering studies aimed at elucidating the physical associations of small molecule ligands with cellular proteins. The technologies covered belong to two main categories: tag-based and tag-free methods. We emphasize the latter in this review, and outline promising experimental workflows and key data analysis considerations involved. Expert opinion: Recent ground-breaking advances in chemical-proteomics technology and allied computational methods now make the global detection of protein-ligand engagement an increasingly attractive research problem. Despite ongoing challenges, rapid progress in the field is expected these coming next few years, leading to a refreshed systems biology research paradigm and much needed new opportunities for improving sparse drug discovery pipelines.

PMID: 28933205 [PubMed - as supplied by publisher]



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NDFIP allows NEDD4/NEDD4L-induced AQP2 ubiquitination and degradation.

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NDFIP allows NEDD4/NEDD4L-induced AQP2 ubiquitination and degradation.

PLoS One. 2017;12(9):e0183774

Authors: Trimpert C, Wesche D, de Groot T, Pimentel Rodriguez MM, Wong V, van den Berg DTM, Cheval L, Ariza CA, Doucet A, Stagljar I, Deen PMT

Abstract
Regulation of our water homeostasis is fine-tuned by dynamic translocation of Aquaporin-2 (AQP2)-bearing vesicles to and from the plasma membrane of renal principal cells. Whereas binding of vasopressin to its type-2 receptor initiates a cAMP-protein kinase A cascade and AQP2 translocation to the apical membrane, this is counteracted by protein kinase C-activating hormones, resulting in ubiquitination-dependent internalization of AQP2. The proteins targeting AQP2 for ubiquitin-mediated degradation are unknown. In collecting duct mpkCCD cells, siRNA knockdown of NEDD4 and NEDD4L E3 ligases yielded increased AQP2 abundance, but they did not bind AQP2. Membrane Yeast Two-Hybrid assays using full-length AQP2 as bait, identified NEDD4 family interacting protein 2 (NDFIP2) to bind AQP2. NDFIP2 and its homologue NDFIP1 have PY motifs by which they bind NEDD4 family members and bring them close to target proteins. In HEK293 cells, NDFIP1 and NDFIP2 bound AQP2 and were essential for NEDD4/NEDD4L-mediated ubiquitination and degradation of AQP2, an effect not observed with PY-lacking NDFIP1/2 proteins. In mpkCCD cells, downregulation of NDFIP1, NEDD4 and NEDD4L, but not NDFIP2, increased AQP2 abundance. In mouse kidney, Ndfip1 and Ndfip2 mRNA distribution was similar and high in proximal tubules and collecting ducts, which was also found for NDFIP1 proteins. Our results reveal that NEDD4/NEDD4L mediate ubiquitination and degradation of AQP2, but that NDFIP proteins are needed to connect NEDD4/NEDD4L to AQP2. As NDFIP1/2 bind many NEDD4 family E3 ligases, which are implicated in several cellular processes, NDFIP1/2 may be the missing link for AQP2 ubiquitination and degradation from different subcellular locations.

PMID: 28931009 [PubMed - in process]



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Protein complexes, big data, machine learning and integrative proteomics: lessons learned over a decade of systematic analysis of protein interaction networks.

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Protein complexes, big data, machine learning and integrative proteomics: lessons learned over a decade of systematic analysis of protein interaction networks.

Expert Rev Proteomics. 2017 Sep 18;:1-11

Authors: Havugimana PC, Hu P, Emili A

Abstract
OVERVIEW: Elucidation of the networks of physical (functional) interactions present in cells and tissues is fundamental for understanding the molecular organization of biological systems, the mechanistic basis of essential and disease-related processes, and for functional annotation of previously uncharacterized proteins (via guilt-by-association or -correlation). After a decade in the field, we felt it timely to document our own experiences in the systematic analysis of protein interaction networks. Areas covered: Researchers worldwide have contributed innovative experimental and computational approaches that have driven the rapidly evolving field of 'functional proteomics'. These include mass spectrometry-based methods to characterize macromolecular complexes on a global-scale and sophisticated data analysis tools - most notably machine learning - that allow for the generation of high-quality protein association maps. Expert commentary: Here, we recount some key lessons learned, with an emphasis on successful workflows, and challenges, arising from our own and other groups' ongoing efforts to generate, interpret and report proteome-scale interaction networks in increasingly diverse biological contexts.

PMID: 28918672 [PubMed - as supplied by publisher]



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P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

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P-body proteins regulate transcriptional rewiring to promote DNA replication stress resistance.

Nat Commun. 2017 Sep 15;8(1):558

Authors: Loll-Krippleber R, Brown GW

Abstract
mRNA-processing (P-) bodies are cytoplasmic granules that form in eukaryotic cells in response to numerous stresses to serve as sites of degradation and storage of mRNAs. Functional P-bodies are critical for the DNA replication stress response in yeast, yet the repertoire of P-body targets and the mechanisms by which P-bodies promote replication stress resistance are unknown. In this study we identify the complete complement of mRNA targets of P-bodies during replication stress induced by hydroxyurea treatment. The key P-body protein Lsm1 controls the abundance of HHT1, ACF4, ARL3, TMA16, RRS1 and YOX1 mRNAs to prevent their toxic accumulation during replication stress. Accumulation of YOX1 mRNA causes aberrant downregulation of a network of genes critical for DNA replication stress resistance and leads to toxic acetaldehyde accumulation. Our data reveal the scope and the targets of regulation by P-body proteins during the DNA replication stress response.P-bodies form in response to stress and act as sites of mRNA storage and degradation. Here the authors identify the mRNA targets of P-bodies during DNA replication stress, and show that P-body proteins act to prevent toxic accumulation of these target transcripts.

PMID: 28916784 [PubMed - in process]



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Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.

Features of the Chaperone Cellular Network Revealed through Systematic Interaction Mapping.

Cell Rep. 2017 Sep 12;20(11):2735-2748

Authors: Rizzolo K, Huen J, Kumar A, Phanse S, Vlasblom J, Kakihara Y, Zeineddine HA, Minic Z, Snider J, Wang W, Pons C, Seraphim TV, Boczek EE, Alberti S, Costanzo M, Myers CL, Stagljar I, Boone C, Babu M, Houry WA

Abstract
A comprehensive view of molecular chaperone function in the cell was obtained through a systematic global integrative network approach based on physical (protein-protein) and genetic (gene-gene or epistatic) interaction mapping. This allowed us to decipher interactions involving all core chaperones (67) and cochaperones (15) of Saccharomyces cerevisiae. Our analysis revealed the presence of a large chaperone functional supercomplex, which we named the naturally joined (NAJ) chaperone complex, encompassing Hsp40, Hsp70, Hsp90, AAA+, CCT, and small Hsps. We further found that many chaperones interact with proteins that form foci or condensates under stress conditions. Using an in vitro reconstitution approach, we demonstrate condensate formation for the highly conserved AAA+ ATPases Rvb1 and Rvb2, which are part of the R2TP complex that interacts with Hsp90. This expanded view of the chaperone network in the cell clearly demonstrates the distinction between chaperones having broad versus narrow substrate specificities in protein homeostasis.

PMID: 28903051 [PubMed - in process]



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State of diagnosing infectious pathogens using colloidal nanomaterials.

State of diagnosing infectious pathogens using colloidal nanomaterials.

Biomaterials. 2017 Aug 17;146:97-114

Authors: Kim J, Mohamed MAA, Zagorovsky K, Chan WCW

Abstract
Infectious diseases are a major global threat that accounts for one of the leading causes of global mortality and morbidity. Prompt diagnosis is a crucial first step in the management of infectious threats, which aims to quarantine infected patients to avoid contacts with healthy individuals and deliver effective treatments prior to further spread of diseases. This review article discusses current advances of diagnostic systems using colloidal nanomaterials (e.g., gold nanoparticles, quantum dots, magnetic nanoparticles) for identifying and differentiating infectious pathogens. The challenges involved in the clinical translation of these emerging nanotechnology based diagnostic devices will also be discussed.

PMID: 28898761 [PubMed - as supplied by publisher]



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The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation.

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The Candida albicans transcription factor Cas5 couples stress responses, drug resistance and cell cycle regulation.

Nat Commun. 2017 Sep 11;8(1):499

Authors: Xie JL, Qin L, Miao Z, Grys BT, Diaz JC, Ting K, Krieger JR, Tong J, Tan K, Leach MD, Ketela T, Moran MF, Krysan DJ, Boone C, Andrews BJ, Selmecki A, Ho Wong K, Robbins N, Cowen LE

Abstract
The capacity to coordinate environmental sensing with initiation of cellular responses underpins microbial survival and is crucial for virulence and stress responses in microbial pathogens. Here we define circuitry that enables the fungal pathogen Candida albicans to couple cell cycle dynamics with responses to cell wall stress induced by echinocandins, a front-line class of antifungal drugs. We discover that the C. albicans transcription factor Cas5 is crucial for proper cell cycle dynamics and responses to echinocandins, which inhibit β-1,3-glucan synthesis. Cas5 has distinct transcriptional targets under basal and stress conditions, is activated by the phosphatase Glc7, and can regulate the expression of target genes in concert with the transcriptional regulators Swi4 and Swi6. Thus, we illuminate a mechanism of transcriptional control that couples cell wall integrity with cell cycle regulation, and uncover circuitry governing antifungal drug resistance.Cas5 is a transcriptional regulator of responses to cell wall stress in the fungal pathogen Candida albicans. Here, Xie et al. show that Cas5 also modulates cell cycle dynamics and responses to antifungal drugs.

PMID: 28894103 [PubMed - in process]



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Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities.

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Comprehensive Analysis of the Human SH3 Domain Family Reveals a Wide Variety of Non-canonical Specificities.

Structure. 2017 Aug 28;:

Authors: Teyra J, Huang H, Jain S, Guan X, Dong A, Liu Y, Tempel W, Min J, Tong Y, Kim PM, Bader GD, Sidhu SS

Abstract
SH3 domains are protein modules that mediate protein-protein interactions in many eukaryotic signal transduction pathways. The majority of SH3 domains studied thus far act by binding to proline-rich sequences in partner proteins, but a growing number of studies have revealed alternative recognition mechanisms. We have comprehensively surveyed the specificity landscape of human SH3 domains in an unbiased manner using peptide-phage display and deep sequencing. Based on ∼70,000 unique binding peptides, we obtained 154 specificity profiles for 115 SH3 domains, which reveal that roughly half of the SH3 domains exhibit non-canonical specificities and collectively recognize a wide variety of peptide motifs, most of which were previously unknown. Crystal structures of SH3 domains with two distinct non-canonical specificities revealed novel peptide-binding modes through an extended surface outside of the canonical proline-binding site. Our results constitute a significant contribution toward a complete understanding of the mechanisms underlying SH3-mediated cellular responses.

PMID: 28890361 [PubMed - as supplied by publisher]



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Variant Interpretation: Functional Assays to the Rescue.

Variant Interpretation: Functional Assays to the Rescue.

Am J Hum Genet. 2017 Sep 07;101(3):315-325

Authors: Starita LM, Ahituv N, Dunham MJ, Kitzman JO, Roth FP, Seelig G, Shendure J, Fowler DM

Abstract
Classical genetic approaches for interpreting variants, such as case-control or co-segregation studies, require finding many individuals with each variant. Because the overwhelming majority of variants are present in only a few living humans, this strategy has clear limits. Fully realizing the clinical potential of genetics requires that we accurately infer pathogenicity even for rare or private variation. Many computational approaches to predicting variant effects have been developed, but they can identify only a small fraction of pathogenic variants with the high confidence that is required in the clinic. Experimentally measuring a variant's functional consequences can provide clearer guidance, but individual assays performed only after the discovery of the variant are both time and resource intensive. Here, we discuss how multiplex assays of variant effect (MAVEs) can be used to measure the functional consequences of all possible variants in disease-relevant loci for a variety of molecular and cellular phenotypes. The resulting large-scale functional data can be combined with machine learning and clinical knowledge for the development of "lookup tables" of accurate pathogenicity predictions. A coordinated effort to produce, analyze, and disseminate large-scale functional data generated by multiplex assays could be essential to addressing the variant-interpretation crisis.

PMID: 28886340 [PubMed - in process]



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Non-base-contacting residues enable kaleidoscopic evolution of metazoan C2H2 zinc finger DNA binding.

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Non-base-contacting residues enable kaleidoscopic evolution of metazoan C2H2 zinc finger DNA binding.

Genome Biol. 2017 Sep 06;18(1):167

Authors: Najafabadi HS, Garton M, Weirauch MT, Mnaimneh S, Yang A, Kim PM, Hughes TR

Abstract
BACKGROUND: The C2H2 zinc finger (C2H2-ZF) is the most numerous protein domain in many metazoans, but is not as frequent or diverse in other eukaryotes. The biochemical and evolutionary mechanisms that underlie the diversity of this DNA-binding domain exclusively in metazoans are, however, mostly unknown.
RESULTS: Here, we show that the C2H2-ZF expansion in metazoans is facilitated by contribution of non-base-contacting residues to DNA binding energy, allowing base-contacting specificity residues to mutate without catastrophic loss of DNA binding. In contrast, C2H2-ZF DNA binding in fungi, plants, and other lineages is constrained by reliance on base-contacting residues for DNA-binding functionality. Reconstructions indicate that virtually every DNA triplet was recognized by at least one C2H2-ZF domain in the common progenitor of placental mammals, but that extant C2H2-ZF domains typically bind different sequences from these ancestral domains, with changes facilitated by non-base-contacting residues.
CONCLUSIONS: Our results suggest that the evolution of C2H2-ZFs in metazoans was expedited by the interaction of non-base-contacting residues with the DNA backbone. We term this phenomenon "kaleidoscopic evolution," to reflect the diversity of both binding motifs and binding motif transitions and the facilitation of their diversification.

PMID: 28877740 [PubMed - in process]



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