Brian T Chait

The Rockefeller University, New York City, New York, United States

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Publications (363)2669.23 Total impact

  • Roman I Subbotin, Brian T Chait
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    ABSTRACT: It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from non-specific interactors, preserving and isolating all unique interactions including those that are weak, transient or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact sub-micron chunks to allow for rapid access of a chemical reagent and to +stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under non-denaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry (SAC-MS). Here, we demonstrate that SAC-MS allows us to stabilize and elucidate local, distant and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.
    Molecular & cellular proteomics : MCP. 08/2014;
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    ABSTRACT: Most cellular processes are orchestrated by macromolecular complexes. However, structural elucidation of these endogenous complexes can be challenging because they frequently contain large numbers of proteins, are compositionally and morphologically heterogeneous, can be dynamic, and are often of low abundance in the cell. Here, we present a strategy for structural characterization of such complexes, which has at its center chemical cross-linking with mass spectrometric readout (CX-MS). In this strategy, we isolate the endogenous complexes using a highly optimized sample preparation protocol and generate a comprehensive, high-quality cross-linking dataset using two complementary cross-linking reagents. We then determine the structure of the complex using a refined integrative method that combines the cross-linking data with information generated from other sources, including electron microscopy, X-ray crystallography, and comparative protein structure modeling. We applied this integrative strategy to determine the structure of the native Nup84 complex , a stable hetero-heptameric assembly (~600 kDa), sixteen copies of which form the outer rings of the 50 MDa nuclear pore complex (NPC) in budding yeast. The unprecedented detail of the Nup84 complex structure reveals previously unseen features in its pentameric structural hub and provides information on the conformational flexibility of the assembly. These additional details further support and augment the protocoatomer hypothesis, which proposes an evolutionary relationship between vesicle coating complexes and the NPC, and indicates a conserved mechanism by which the NPC is anchored in the nuclear envelope.
    Molecular & cellular proteomics : MCP. 08/2014;
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    ABSTRACT: Autophagy is a tightly regulated lysosomal degradation pathway for maintaining cellular homeostasis and responding to stresses. Beclin 1 and its interacting proteins, including the class III phosphatidylinositol-3 kinase Vps34, play crucial roles in autophagy regulation in mammals. We identified nuclear receptor binding factor 2 (Nrbf2) as a Beclin 1-interacting protein from Becn1-/-;Becn1-EGFP/+ mouse liver and brain. We also found that Nrbf2-Beclin 1 interaction required the N-terminus of Nrbf2. We next used human retinal pigment epithelial cell line RPE-1 as a model system and showed that transiently knocking down Nrbf2 by siRNA increased autophagic flux under both nutrient-rich and starvation conditions. To investigate the mechanism by which Nrbf2 regulates autophagy, we demonstrated that Nrbf2 interacted and colocalized with Atg14L, suggesting that Nrbf2 is a component of the Atg14L-containing Beclin 1-Vps34 complex. Moreover, ectopically expressed Nrbf2 formed cytosolic puncta that were positive for isolation membrane markers. These results suggest that Nrbf2 is involved in autophagosome biogenesis. Furthermore, we showed that Nrbf2 deficiency led to increased intracellular phosphatidylinositol-3 phosphate levels as well as diminished Atg14L-Vps34/Vps15 interactions, suggesting that Nrbf2-mediated Atg14L-Vps34/Vps15 interactions likely inhibit Vps34 activity. Therefore, we propose that Nrbf2 may interact with the Atg14L-containing Beclin 1-Vps34 protein complex to modulate protein-protein interactions within the network, leading to suppression of Vps34 activity, autophagosome biogenesis and autophagic flux. This work reveals a novel aspect of the intricate mechanism for the Beclin 1-Vps34 protein-protein interaction network to achieve precise control of autophagy.
    The Journal of biological chemistry. 08/2014;
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    ABSTRACT: The nuclear pore complex (NPC) is the sole mediator of bidirectional nucleo-cytoplasmic transport and is also an important scaffold for chromatin organization and transcriptional regulation. Proteomic studies of numerous diverse eukaryotic species initially characterized the NPC as built with a number of remarkably similar structural features, suggesting its status as an ancient and conserved eukaryotic cell component. However, further detailed analyses now suggest that several key specific NPC features have a more convoluted evolutionary history than initially assumed. Recently we reported on TbNup92, a component in trypanosomes of one such conserved structural feature, a basket-like structure on the nuclear face of the NPC. We showed that TbNup92 has similar roles to nuclear basket proteins from yeasts and animals (Mlp and Tpr, respectively) in interacting with both the NPC and the mitotic spindle. However, comparative genomics suggests that TbNup92 and Mlp/Tpr may be products of distinct evolutionary histories, raising the possibility that these gene products are analogs rather than direct orthologs. Taken together with recent evidence for divergence in the nuclear lamina and kinetochores, it is apparent that the trypanosome nucleus functions by employing several novel or highly divergent protein complexes in parallel with conserved elements. These findings have major implications for how the trypanosomatid nucleus operates and the evolution of hierarchical nuclear organization.
    Nucleus (Austin, Tex.). 07/2014; 5(4).
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    ABSTRACT: The TORC1 signaling pathway plays a major role in the control of cell growth and response to stress. Here we demonstrate that the SEA complex physically interacts with TORC1 and is an important regulator of its activity. During nitrogen starvation, deletions of SEA complex components lead to Tor1 kinase delocalization, defects in autophagy and vacuolar fragmentation. TORC1 inactivation, via nitrogen deprivation or rapamycin treatment, changes cellular level of SEA complex members. We used affinity purification and chemical cross-linking to generate the data for an integrative structure modeling approach, which produced a well-defined molecular architecture of the SEA complex and showed that the SEA complex to be comprised of two regions that are structurally and functionally distinct. The SEA complex emerges as a platform that can coordinate both structural and enzymatic activities necessary for the effective functioning of the TORC1 pathway.
    Molecular & cellular proteomics : MCP. 07/2014;
  • Yinyin Li, Frederick R Cross, Brian T Chait
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    ABSTRACT: In eukaryotes, cell cycle progression is controlled by cyclin/cyclin-dependent kinase (CDK) pairs. To better understand the details of this process, it is necessary to dissect the CDK's substrate pool in a cyclin- and cell cycle stage-specific way. Here, we report a mass spectrometry-based method that couples rapid isolation of native kinase-substrate complexes to on-bead phosphorylation with heavy-labeled ATP (ATP-γ-(18)O4). This combined in vivo/in vitro method was developed for identifying cyclin/CDK substrates together with their sites of phosphorylation. We used the method to identify Clb5 (S-cyclin)/Cdc28 and Cln2 (G1/S-cyclin)/Cdc28 substrates during S phase in Saccharomyces cerevisiae (Cdc28 is the master CDK in budding yeast). During the work, we discovered that Clb5/Cdc28 specifically phosphorylates S429 in the disordered tail of Cdc14, an essential phosphatase antagonist of Cdc28. This phosphorylation severely decreases the activity of Cdc14, providing a means for modulating the balance of CDK and phosphatase activity.
    Proceedings of the National Academy of Sciences of the United States of America. 07/2014;
  • Brian Chait
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    Journal of the American Society for Mass Spectrometry 03/2014; · 3.59 Impact Factor
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    ABSTRACT: The nuclear pore complex (NPC) has dual roles in nucleocytoplasmic transport and chromatin organisation. In many eukaryotes the coiled-coil Mlp/Tpr proteins of the NPC nuclear basket have specific functions in interactions with chromatin and defining specialised regions of active transcription, while Mlp2 associates with the mitotic spindle/NPC in a cell cycle-dependent manner. We previously identified two putative Mlp-related proteins in African trypanosomes, TbNup110 and TbNup92, the latter of which associates with the spindle. We now provide evidence for independent ancestry for TbNup92/TbNup110 and Mlp/Tpr proteins. However, TbNup92 is required for correct chromosome segregation, with knockout cells exhibiting microaneuploidy and lowered fidelity telomere segregation. Further, TbNup92 is intimately associated with the mitotic spindle and spindle anchor site, but apparently has minimal roles in control of gene transcription, indicating that TbNup92 lacks major barrier activity. TbNup92 therefore acts as a functional analog of Mlp/Tpr proteins, and together with the lamina analog NUP-1, represents a cohort of novel proteins operating at the nuclear periphery of trypanosomes, uncovering complex evolutionary trajectories for the NPC and nuclear lamina.
    Molecular biology of the cell 03/2014; · 5.98 Impact Factor
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    ABSTRACT: In vitro mimics of cellular machines have been recently engineered and utilized to investigate processes within cells. These devices can provide novel insights into biological mechanisms and have the potential to improve biotechnological processes such as separation. In particular, several devices have been designed to mimic translocation through nuclear pore complexes (NPCs). We describe here the fabrication of a biomimetic NPC using nanoporous filters lined with FG-repeats of proteins that create a selectivity barrier. We show the utility of this nanoselective filter as a testbed for the investigation of nucleocytoplasmic transport and demonstrate that this device closely reproduces key features of trafficking through the NPC.
    Methods in cell biology 01/2014; 122C:379-393. · 1.44 Impact Factor
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    ABSTRACT: Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.
    PLoS ONE 01/2014; 9(4):e94122. · 3.73 Impact Factor
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    ABSTRACT: LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their life cycles, whereas hosts have developed mechanisms to combat retrotransposition's mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here, we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the copurified proteome, identifying 37 high-confidence candidate interactors. These data sets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest that this occurs during or immediately after target-primed reverse transcription.
    Cell 11/2013; 155(5):1034-1048. · 31.96 Impact Factor
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    ABSTRACT: The basket of the nuclear pore complex (NPC) is generally depicted as a discrete structure of eight protein filaments that protrude into the nucleoplasm and converge in a ring distal to the NPC. We show that the yeast proteins Mlp1p and Mlp2p are necessary components of the nuclear basket and that they also embed the NPC within a dynamic protein network, whose extended interactome includes the spindle organizer, silencing factors, the proteasome, and key components of messenger RNPs (mRNPs). Ultrastructural observations indicate that the basket reduces chromatin crowding around the central transporter of the NPC and might function as a docking site for mRNP during nuclear export. In addition, we show that the Mlps contribute to NPC positioning, nuclear stability, and NE morphology. Our results suggest that the Mlps are multifunctional proteins linking the nuclear transport channel to multiple macromolecular complexes involved in the regulation of gene expression and chromatin maintenance.
    Molecular biology of the cell 10/2013; · 5.98 Impact Factor
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    ABSTRACT: Cytokinesis, the process in which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II-binding proteins anillin and supervillin, act earlier. Anillin's role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831-1281, which bind central spindle proteins, and residues 1-170, which bind the myosin II heavy chain (MHC) and the long form of myosin light chain kinase (l-MLCK). MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increase cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity GFP nanobody suggest that supervillin and anillin regulate the myosin II- and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.
    Molecular biology of the cell 10/2013; · 5.98 Impact Factor
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    ABSTRACT: Nuclear shape and size vary between species, during development, and in many tissue pathologies, but the causes and effects of these differences remain poorly understood. During fertilization, sperm nuclei undergo a dramatic conversion from a heavily compacted form into decondensed, spherical pronuclei, accompanied by rapid nucleation of microtubules from centrosomes. Here we report that the assembly of the spherical nucleus depends on a critical balance of microtubule dynamics, which is regulated by the chromatin-binding protein Developmental pluripotency-associated 2 (Dppa2). Whereas microtubules normally promote sperm pronuclear expansion, in Dppa2-depleted Xenopus egg extracts excess microtubules cause pronuclear assembly defects, leading to abnormal morphology and disorganized DNA replication. Dppa2 inhibits microtubule polymerization in vitro, and Dppa2 activity is needed at a precise time and location during nascent pronuclear formation. This demonstrates a strict spatiotemporal requirement for local suppression of microtubules during nuclear formation, fulfilled by chromatin-bound microtubule regulators.
    Developmental Cell 09/2013; 27(1):47-59. · 12.86 Impact Factor
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    ABSTRACT: Regulation of gene expression in a vital part of the cellular stress response, yet the full set of proteins that orchestrate this regulation remains unknown. Snt2 is a yeast protein whose function has not been well characterized, that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Here, we confirm that Snt2, Ecm5, and Rpd3 physically associate. We then demonstrate that cells lacking Rpd3 or Snt2 are resistant to hydrogen peroxide (H2O2)-mediated oxidative stress, and use chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) to show that Snt2 and Ecm5 recruit Rpd3 to a small number of promoters and in response to H2O2, colocalize independent of Rpd3 to promoters of stress response genes. By integrating ChIP-seq and expression analysis, we identify target genes that require Snt2 for proper expression after H2O2. Finally, we show that cells lacking Snt2 are also resistant to nutrient stress imparted by the TOR pathway inhibitor rapamycin, and identify a common set of genes targeted by Snt2 and Ecm5 in response to both H2O2 and rapamycin. Our results establish a function for Snt2 in regulating transcription in response to oxidative stress, and suggest Snt2 may also function in multiple stress pathways.
    Molecular and cellular biology 07/2013; · 6.06 Impact Factor
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    ABSTRACT: Selected Reaction Monitoring (SRM) is a method of choice for accurate quantitation of low-abundance proteins in complex backgrounds. This strategy is, however, sensitive to interference from other componentsin the sample that have the same precursor and fragment masses as the monitored transitions. We present here an approach to detect interference by using the expected relative intensity of SRM transitions. We also designed an algorithm to automatically detect the linear range of calibration curves. These approaches were applied to the experimental data of Clinical Proteomic Tumor Analysis Consortium (CPTAC) Verification Work Group Study 7 and show that the corrected measurements provide more accurate quantitation than the uncorrected data.
    Methods 05/2013; · 3.64 Impact Factor
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    ABSTRACT: p11, through unknown mechanisms, is required for behavioral and cellular responses to selective serotonin reuptake inhibitors (SSRIs). We show that SMARCA3, a chromatin-remodeling factor, is a target for the p11/annexin A2 heterotetrameric complex. Determination of the crystal structure indicates that SMARCA3 peptide binds to a hydrophobic pocket in the heterotetramer. Formation of this complex increases the DNA-binding affinity of SMARCA3 and its localization to the nuclear matrix fraction. In the dentate gyrus, both p11 and SMARCA3 are highly enriched in hilar mossy cells and basket cells. The SSRI fluoxetine induces expression of p11 in both cell types and increases the amount of the ternary complex of p11/annexin A2/SMARCA3. SSRI-induced neurogenesis and behavioral responses are abolished by constitutive knockout of SMARCA3. Our studies indicate a central role for a chromatin-remodeling factor in the SSRI/p11 signaling pathway and suggest an approach to the development of improved antidepressant therapies. PAPERCLIP:
    Cell 02/2013; 152(4):831-43. · 31.96 Impact Factor
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    ABSTRACT: DNA double-strand breaks (DSBs) represent a threat to the genome because they can lead to loss of genetic information and chromosome rearrangements. The DNA repair protein p53 binding protein 1 (53BP1) protects the genome by limiting nucleolytic processing of DSBs by a mechanism that requires its phosphorylation, but whether it does so directly is not known. Here, we identify Rapl-interacting factor 1 (Rif1) as an Ataxia-Telangiectasia Mutated (ATM) phosphorylation-dependent interactor of 53BP1, and show that absence of Rif1 results in 5'-3' DNA end resection in mice. Consistent with enhanced DNA resection, Rif1 deficiency impairs DNA repair in the G1 and S phases of the cell cycle, interferes with class switch recombination in B lymphocytes, and leads to accumulation of chromosome DSBs.
    Science 01/2013; · 31.20 Impact Factor

Publication Stats

27k Citations
2,669.23 Total Impact Points

Institutions

  • 1981–2014
    • The Rockefeller University
      • • Laboratory of Mass Spectrometry and Gaseous Ion Chemistry
      • • Laboratory of Molecular Immunology
      • • Laboratory of the Biology of Addictive Diseases
      • • Laboratory of Cellular and Structural Biology
      • • Laboratory of Biochemistry and Molecular Biology
      • • Laboratory of Molecular Neurobiology and Biophysics
      New York City, New York, United States
  • 2012
    • University of Cambridge
      • Department of Pathology
      Cambridge, ENG, United Kingdom
  • 2011
    • French National Centre for Scientific Research
      • Institut Jacques-Monod
      Paris, Ile-de-France, France
  • 2007–2011
    • Mount Sinai School of Medicine
      • Department of Neuroscience
      Manhattan, NY, United States
  • 1998–2010
    • Northeastern University
      • Department of Biology
      Boston, MA, United States
    • Cornell University
      • Department of Pediatrics
      Ithaca, NY, United States
  • 2008
    • Princeton University
      • Department of Molecular Biology
      Princeton, NJ, United States
    • University of Birmingham
      • School of Biosciences
      Birmingham, ENG, United Kingdom
  • 2006–2007
    • University of California, San Francisco
      • • Department of Pharmaceutical Chemistry
      • • Department of Bioengineering and Therapeutic Sciences
      San Francisco, CA, United States
    • Howard Hughes Medical Institute
      Maryland, United States
  • 2005
    • The California Institute for Biomedical Research
      San Diego, California, United States
  • 2000
    • Okayama University
      Okayama, Okayama, Japan
    • University Hospital Frankfurt
      Frankfurt, Hesse, Germany
  • 1987–1996
    • CUNY Graduate Center
      New York City, New York, United States
  • 1992
    • Carnegie Mellon University
      • Department of Chemistry
      Pittsburgh, PA, United States
    • University of Münster
      Muenster, North Rhine-Westphalia, Germany
  • 1989–1992
    • University of California, Irvine
      • Department of Biological Chemistry
      Irvine, CA, United States
    • Dalhousie University
      Halifax, Nova Scotia, Canada
  • 1981–1987
    • University of Manitoba
      • Department of Chemistry
      Winnipeg, Manitoba, Canada