No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Integrative Modeling of a Sin3/HDAC Complex Sub-Structure
... BRMS1 is a known member of Sin3 histone deacetylase (HDAC) transcriptional regulatory complexes in multiple eukaryotic cell types [11][12][13]. It is an established binding partner of SUDS3 and ARID4A and is also associated with several other members of the complex such as SIN3A, SIN3B, HDAC1, and HDAC2 [11]. ...
... Utilizing recently published crosslinking data of Sin3/HDAC complex members [12], BRMS1 was confirmed to interact with its known interaction partner SUDS3 (at BRMS1 aa 142), while also binding to the Sin3/HDAC member HDAC1 at BRMS1 aa 184 ( Figure 1C). BRMS1 also cross-links with SIN3A but not with the related SIN3B ( Figure 1C). ...
... A short synthetic duplex DNA oligonucleotide (described in Supplementary Data) was used to clone a short fragment of the C terminus of BRMS1 (amino acids 230-246). DNA fragments were digested with SgfI and PmeI and inserted between SgfI and PmeI sites in pcDNA5FRT-Halo [12]. ...
Breast Cancer Metastasis Suppressor 1 (BRMS1) expression has been associated with longer patient survival in multiple cancer types. Understanding BRMS1 at the protein level will provide insights into both mechanism of action and enhance potential therapeutic development. We previously mapped the C-terminus of BRMS1 as critical for metastasis suppression and hypothesized that critical protein interactions in this region will explain function. These studies indicate that phosphorylation status at S237 regulates BRMS1 interactions related to a variety of biological processes, phenotypes [cell cycle (e.g., CDKN2A), DNA repair (e.g., BRCA1)], and metastasis [(e.g., TCF2 and POLE2)]. Presence of the C-terminal site appears to be critical for BRMS1 directed metastasis suppression, as demonstrated by in vitro migration assays. These assays demonstrated that presence of S237 directly decreased MDA-MB-231 migration. This study furthers our understanding of BRMS1’s molecular role, as it demonstrates that BRMS1 C-terminus is involved in direct protein-protein interactions. Several of the interacting proteins are associated with cancer and metastasis, which may result in metastasis suppression as suggested by in vitro findings.
Abstract Figure
Graphical Abstract
Utilizing BRMS1 mutants to mimic-phosphorylation, this study demonstrates that S237-phosphorylation disrupts BRMS1 protein-protein interactions. The disruption includes both known Sin3/HDAC interactors as well as additionally previously unidentified Sin3-indepedent binding partners (indicated by increased opacity). It is revealed that BRMS1-phosphorylation status also more greatly inhibits cell migration (indicated by +) compared to the unphosphorylated state, suggesting that phosphorylation plays a role in BRMS1 metastatsis suppresion function, potentially though altered protein interactions.
... The full suite of identified crosslinks is depicted as a circos plot in Fig. 2A, while the identified C. reinhardtii interactome is displayed in Fig. 2B (interactive versions of both are located at https://tinyurl.com/HicksXLMS). The majority of the CSMs are contained in later SCX fractions, [49][50][51][52][53][54][55][56][57][58][59][60][61][62][63][64][65] supporting the use of SCX as an enrichment technique for more positively charged crosslinked peptides (Fig. 2C). Among the 769 detected crosslinks, 157 are between two tryptic peptides derived from two different proteins (interlinks) and 612 are between two tryptic peptides within the same protein (intralinks) (Fig. 2D). ...
... Overlaying detected intralinks onto known protein structures and comparing the Euclidean distance between the crosslinked residues to the DSSO maximum crosslinking distance of 30 Å can be used to evaluate the intralink dataset. 13,[50][51][52] Few proteins with identified intralinks have existing structures for C. reinhardtii in the Protein Data Bank (PDB), so the Integrative Modeling Platform in XLinkDB 3.0 was used for homology modeling for the other proteins. 44,46 In this platform, a structural homology model is identified by multiple sequence alignment and used to predict protein structure. ...
Interactomics is an emerging field that seeks to identify both transient and complex-bound protein interactions that are essential for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled untargeted global analysis of these protein networks, permitting largescale simultaneous analysis of protein structure and interactions. Increased commercial availability of highly specific, cell permeable crosslinkers has propelled the study of these critical interactions forward, with the development of MS-cleavable crosslinkers further increasing confidence in identifications. Herein, the global interactome of the unicellular alga Chlamydomonas reinhardtii was analyzed via XL-MS by implementing the MS-cleavable disuccinimidyl sulfoxide (DSSO) crosslinker and enriching for crosslinks using strong cation exchange chromatography. Gentle lysis via repeated freeze–thaw cycles facilitated in vitro analysis of 157 protein–protein crosslinks (interlinks) and 612 peptides linked to peptides of the same protein (intralinks) at 1% FDR throughout the C. reinhardtii proteome. The interlinks confirmed known protein relationships across the cytosol and chloroplast, including coverage on 42% and 38% of the small and large ribosomal subunits, respectively. Of the 157 identified interlinks, 92% represent the first empirical evidence of interaction observed in C. reinhardtii. Several of these crosslinks point to novel associations between proteins, including the identification of a previously uncharacterized Mg-chelatase associated protein (Cre11.g477733.t1.2) bound to seven distinct lysines on Mg-chelatase (Cre06.g306300.t1.2). Additionally, the observed intralinks facilitated characterization of novel protein structures across the C. reinhardtii proteome. Together, these data establish a framework of protein–protein interactions that can be further explored to facilitate understanding of the dynamic protein landscape in C. reinhardtii.
... However, these structural and biochemical studies focused only on the major determinants of these interactions; our cryo-EM structure reveals additional interactions governing the higher-order folding of the complex, thereby providing unprecedented insights into how the complex is assembled and organized. The overall architecture of the yeast complex is also broadly consistent with previous crosslinking mass spectrometric analyses of the mammalian complex, thereby providing additional independent support for our structure 36 . Previous studies anticipated a symmetric complex based on the presence of multiple dimerization motifs 27 ; however, as vividly illustrated in our structure, these motifs mediate heterodimerization and perform important scaffolding functions for assembly and higher-order folding of the complex (Fig. 2B). ...
The Rpd3L histone deacetylase (HDAC) complex is an ancient 12-subunit complex conserved in a broad range of eukaryotes that performs localized deacetylation at or near sites of recruitment by DNA-bound factors. Here we describe the cryo-EM structure of this prototypical HDAC complex that is characterized by as many as seven subunits performing scaffolding roles for the tight integration of the only catalytic subunit, Rpd3. The principal scaffolding protein, Sin3, along with Rpd3 and the histone chaperone, Ume1, are present in two copies, with each copy organized into separate lobes of an asymmetric dimeric molecular assembly. The active site of one Rpd3 is completely occluded by a leucine side chain of Rxt2, while the tips of the two lobes and the more peripherally associated subunits exhibit varying levels of flexibility and positional disorder. The structure reveals unexpected structural homology/analogy between unrelated subunits in the fungal and mammalian complexes and provides a foundation for deeper interrogations of structure, biology, and mechanism of these complexes, as well as for the discovery of HDAC complex-specific inhibitors.
... The highly conserved, multidomain-containing protein Sin3 is thought to provide a platform for the assembly of HDACs and non-catalytic subunits, forming two major types of the SIN3/HDAC complexes (SIN3L and SIN3S) [7][8][9][10] . The SIN3L complex, consisting of 10-15 protein components with a combined molecular weight of 1 megadaltons (MDa), is broadly recruited to the promoter of target genes through DNA-binding factors or other corepressors to inhibit transcription [11][12][13][14] . Moreover, the~0.6 ...
The switch-independent 3 (SIN3)/histone deacetylase (HDAC) complexes play essential roles in regulating chromatin accessibility and gene expression. There are two major types of SIN3/HDAC complexes (named SIN3L and SIN3S) targeting different chromatin regions. Here we present the cryo-electron microscopy structures of the SIN3L and SIN3S complexes from Schizosaccharomyces pombe (S. pombe), revealing two distinct assembly modes. In the structure of SIN3L, each Sin3 isoform (Pst1 and Pst3) interacts with one histone deacetylase Clr6, and one WD40-containing protein Prw1, forming two lobes. These two lobes are bridged by two vertical coiled-coil domains from Sds3/Dep1 and Rxt2/Png2, respectively. In the structure of SIN3S, there is only one lobe organized by another Sin3 isoform Pst2; each of the Cph1 and Cph2 binds to an Eaf3 molecule, providing two modules for histone recognition and binding. Notably, the Pst1 Lobe in SIN3L and the Pst2 Lobe in SIN3S adopt similar conformation with their deacetylase active sites exposed to the space; however, the Pst3 Lobe in SIN3L is in a compact state with its active center buried inside and blocked. Our work reveals two classical organization mechanisms for the SIN3/HDAC complexes to achieve specific targeting and provides a framework for studying the histone deacetylase complexes.
... As self-association is a fairly common physical property of proteins [41], HDAC6 oligomerization can, in principle, confer general functional advantages such as increased stability, increased substrate affinity by avidity effect, facilitating pre-concentration of molecules, and generating links between different cellular components. It is interesting to note that while not homodimerizing, zinc-dependent class I HDACs are typically components of large corepressor complexes that define their target selectivity [42][43][44][45][46][47] and HDAC heterodimerization regulates their deacetylase activity [48]. ...
Human histone deacetylase 6 (HDAC6) is a structurally unique, multidomain protein implicated in a variety of physiological processes including cytoskeletal remodelling and the maintenance of cellular homeostasis. Our current understanding of the HDAC6 structure is limited to isolated domains, and a holistic picture of the full‐length protein structure, including possible domain interactions, is missing. Here, we used an integrative structural biology approach to build a solution model of HDAC6 by combining experimental data from several orthogonal biophysical techniques complemented by molecular modelling. We show that HDAC6 is best described as a mosaic of folded and intrinsically disordered domains that in‐solution adopts an ensemble of conformations without any stable interactions between structured domains. Furthermore, HDAC6 forms dimers/higher oligomers in a concentration‐dependent manner, and its oligomerization is mediated via the positively charged N‐terminal microtubule‐binding domain. Our findings provide the first insights into the structure of full‐length human HDAC6 and can be used as a basis for further research into structure function and physiological studies of this unique deacetylase.
... Cell-type-specific regulation of protein complex dynamics is one potential disease-modifying factor. A single protein can participate in different biological processes as a component of distinct protein complexes (Antonicka et al., 2020;Banks et al., 2020;Joshi et al., 2013). These functional protein assemblies can be spatially and temporally regulated within a cell and may contain numerous protein interactions with a range of dynamic properties from stable to transient (Budayeva and Cristea, 2014;Hashimoto et al., 2020;Liu et al., 2016;Przytycka et al., 2010). ...
Huntington disease (HD) is a monogenic neurodegenerative disorder with one causative gene, huntingtin (HTT). Yet, HD pathobiology is multifactorial, suggesting that cellular factors influence disease progression. Here, we define HTT protein-protein interactions (PPIs) perturbed by the mutant protein with expanded polyglutamine in the mouse striatum, a brain region with selective HD vulnerability. Using metabolically labeled tissues and immunoaffinity purification-mass spectrometry, we establish that polyglutamine-dependent modulation of HTT PPI abundances and relative stability starts at an early stage of pathogenesis in a Q140 HD mouse model. We identify direct and indirect PPIs that are also genetic disease modifiers using in-cell two-hybrid and behavioral assays in HD human cell and Drosophila models, respectively. Validated, disease-relevant mHTT-dependent interactions encompass mediators of synaptic neurotransmission (SNAREs and glutamate receptors) and lysosomal acidification (V-ATPase). Our study provides a resource for understanding mHTT-dependent dysfunction in cortico-striatal cellular networks, partly through impaired synaptic communication and endosomal-lysosomal system. A record of this paper’s Transparent Peer Review process is included in the supplemental information.
... However, in the present work, we failed to observe any transcriptional differences of class I HDACs (HDAC1, HDAC2, and HDAC3) in mice treated with repeated dose of vehicle or oxaliplatin. However, several genes encoding for methyl transferase (Setd1b, Mettl8, kmt2d) were found to be downregulated, and genes associated to histone deacetylase complex such as Sap30l [29] and Banp [30] were upregulated in DRG neurons from oxaliplatin-treated animals (see supplementary data S1). As DRGs are a heterogeneous tissue containing the cell bodies of peripheral nerves, epithelial cells, fibroblasts, glial cells, and immune cells, DEGs discovered following treatment with oxaliplatin cannot be unequivocally assigned to sensory neurons alone. ...
Oxaliplatin, the first-line chemotherapeutic agent against colorectal cancer (CRC), induces peripheral neuropathies, which can lead to dose limitation and treatment discontinuation. Downregulation of potassium channels, which involves histone deacetylase (HDAC) activity, has been identified as an important tuner of acute oxaliplatin-induced hypersensitivity. MS-275, a class I histone deacetylase inhibitor (HDACi), prevents acute oxaliplatin-induced peripheral neuropathy (OIPN). Moreover, MS-275 exerts anti-tumor activity in several types of cancers, including CRC. We thus hypothesized that MS-275 could exert both a preventive effect against OIPN and potentially a synergistic effect combined with oxaliplatin against CRC development. We first used RNAseq to assess transcriptional changes occurring in DRG neurons from mice treated by repeated injection of oxaliplatin. Moreover, we assessed the effects of MS-275 on chronic oxaliplatin-induced peripheral neuropathy development in vivo on APC Min/+ mice and on cancer progression when combined with oxaliplatin, both in vivo on APC Min/+ mice and in a mouse model of an orthotopic allograft of the CT26 cell line as well as in vitro in T84 and HT29 human CRC cell lines. We found 741 differentially expressed genes (DEGs) between oxaliplatin-and vehicle-treated animals. While acute OIPN is known as a channelopathy involving HDAC activity, chronic OIPN exerts weak ion channel transcriptional changes and no HDAC expression changes in peripheral neurons from OIPN mice. However, MS-275 prevents the development of sensory neuropathic symptoms induced by repeated oxaliplatin administration in APC Min/+ mice. Moreover, combined with oxaliplatin, MS-275 also exerts synergistic antiproliferative and increased survival effects in CT26-bearing mice. Consistently, combined drug associations exert synergic apoptotic and cell death effects in both T84 and HT29 human CRC cell lines. Our results strongly suggest combining oxaliplatin and MS-275 administration in CRC patients in order to potentiate the antiproliferative action of chemotherapy, while preventing its neurotoxic effect. Citation: Lamoine, S.; Cumenal, M.; Barriere, D.A.; Pereira, V.; Fereyrolles, M.; Prival, L.; Barbier, J.; Boudieu, L.; Brasset, E.; Bertin, B.; et al. The Class I HDAC Inhibitor, MS-275, Prevents Oxaliplatin-Induced Chronic Neuropathy and Potentiates Its Antiproliferative Activity in Mice. Int. J. Mol. Sci. 2022, 23, 98.
... On the one hand Stb1 could act as repressor by promoting repressive function of the Sin3/Rpd3-complex, and on the other hand act as activator by mediating Sin3/Rpd3 inactivation and its dissociation from Swi6 (Fig. 2D). Such a model postulates complex sub-structures and conformational changes within the Sin3/Rpd3-complex what corresponds to the results of Banks et al. who show that multiple interface points and protein subunits specifically organize around the scaffold Sin3 to facilitate catalytic function of the assembly (Banks et al. 2020). Ho et al. and Costanzo et al. demonstrate via in vitro kinase assays that Stb1 is downstream target specifically of Cln1-Cdc28 and Cln2-Cdc28 kinases (Ho et al. 1999;Costanzo, Schub and Andrews 2003), and thus they conclude that both G1-related Cyclin-CDK complexes are the physiological kinases of Stb1. ...
Molecular signal transduction networks which conduct transcription at the G1 to S phase transition of the eukaryotic cell division cycle have been identified in diverse taxa from mammals to baker´s yeast with analogous functional organization. However, regarding some network components, such as the transcriptional regulators STB1 and WHI5, only few orthologs exist which are confined to individual Saccharomycotina species. While Whi5 has been characterized as yeast analog of human Rb protein, in the particular case of Stb1 (Sin three binding protein 1) identification of functional analogs emerges as difficult because to date its exact functionality still remains obscured. By aiming to resolve Stb1´s enigmatic role this Perspectives article especially surveys works covering relations between Cyclin/CDKs, the heteromeric transcription factor complexes SBF (Swi4/Swi6) and MBF (Mbp1/Swi6), as well as additional coregulators (Whi5, Sin3, Rpd3, Nrm1) which are collectively associated with the orderly transcription at 'Start' of the Saccharomyces cerevisiae cell cycle. In this context, interaction capacities of the Sin3-scaffold protein are widely surveyed because its four PAH domains (Paired Amphiphatic Helix) represent a 'recruitment-code' for gene-specific targeting of repressive histone deacetylase activity (Rpd3) via different transcription factors. Here Stb1 plays a role in Sin3´s action on transcription at the G1/S-boundary. Through bioinformatic analyses a potential Sin3-interaction domain (SID) was detected in Stb1, and beyond that, connections within the G1/S-regulatory network are discussed in structural and evolutionary context thereby providing conceptual perspectives.
... The 1.2-2 MDa mammalian complex harbors at least 10 constitutively associated subunits including Sin3A/B, HDAC1/2, RBBP4/7, Sds3/BRMS1/BRMS1L, SAP30/SAP30L, ING1b/ING2, SAP130a/b, ARID4A/B, FAM60A, and SAP25 (paralogous proteins in this list are separated by a '/'). The first five subunits on the list comprise the core complex because of their essential roles in complex assembly and stability (16)(17)(18)(19); these subunits along with the ING subunits have orthologs in yeast. Whereas the RBBP, ING, and ARID4 subunits harbor WD-40, PHD, and Royal Family domains that bind unmodified and modified histones, the other subunits of the complex harbor conserved domains of unknown structure and function. ...
Chromatin-modifying complexes containing histone deacetylase (HDAC) activities play critical roles in the regulation of gene transcription in eukaryotes. These complexes are thought to lack intrinsic DNA-binding activity, but according to a well-established paradigm, they are recruited via protein-protein interactions by gene-specific transcription factors and post-translational histone modifications to their sites of action on the genome. The mammalian Sin3L/Rpd3L complex, comprising more than a dozen different polypeptides, is an ancient HDAC complex found in diverse eukaryotes. The subunits of this complex harbor conserved domains and motifs of unknown structure and function. Here we show that Sds3, a constitutively associated subunit critical for the proper functioning of the complex, harbors a type of Tudor domain that we designate the capped Tudor domain (CTD). Unlike canonical Tudor domains that bind modified histones, the Sds3 CTD binds to nucleic acids that can form higher-order structures such as G-quadruplexes, and shares similarities with the knotted Tudor domain of the Esa1 histone acetyltransferase (HAT) that was previously shown to bind single-stranded RNA. Our findings expand the range of macromolecules capable of recruiting the Sin3L/Rpd3L complex and draws attention to potentially new roles for this HDAC complex in transcription biology.
... In addition, this study found that HDAC1 inhibition alone had little effect on the acetylation of FoxO1, which may be related to the fact that the deacetylation of HDAC1 requires the cooperation of the nuclear cofactor Ski. 33 The present study found a significant reduction in Ski expression under HG conditions, which provided the basis for HDAC1's inability to perform deacetylation. ...
Objectives
The present study clarified the role and signalling pathway of Ski in regulating proliferation and apoptosis in fibroblasts under high‐glucose (HG) conditions.
Materials and Methods
The proliferation and apoptosis of rat primary fibroblasts were assessed using EdU incorporation and TUNEL assays. The protein and phosphorylation levels of the corresponding factors were measured using immunofluorescence staining and Western blotting. Immunoprecipitation was used to determine the interactions between Ski and FoxO1 or Ski and HDAC1. The Ski protein was overexpressed via recombinant adenovirus transfection, and FoxO1 and HDAC1 were knocked down using targeted small‐interfering RNA.
Results
The present study found that HG inhibited fibroblast proliferation, increased apoptosis and reduced Ski levels in rat primary fibroblasts. Conversely, increasing Ski protein levels alleviated HG‐induced proliferation inhibition and apoptosis promotion. Increasing Ski protein levels also increased Ski binding to FoxO1 to decrease FoxO1 acetylation, and interfering with FoxO1 caused loss of the regulatory effect of Ski in fibroblasts under HG. Increasing Ski protein levels decreased FoxO1 acetylation via HDAC1‐mediated deacetylation.
Conclusions
Therefore, these findings confirmed for the first time that Ski regulated fibroblast proliferation and apoptosis under HG conditions via the FoxO1 pathway.
... In principle, the approach could be extended to prepare samples of larger protein complexes for analysis and structural modeling by tagging two components of a larger multisubunit complex. Our recent study modeling a substructure of the relatively abundant chromatin remodeler Sin3 suggests that this would be feasible (48). Although we were able to model a three-subunit substructure using a single-step Halo affinity purification in this case, the improvements in sample preparation offered by the SCAP approach might make similar studies with less abundant larger complexes more feasible. ...
Significance
Determining the three-dimensional structures of protein complexes is critically important to guide biological research. Structural models of complexes can be built using powerful integrative approaches that combine emerging technologies in mass spectrometry, molecular modeling, and protein docking; however, preparing enriched biochemical samples suitable for analysis remains a major challenge. Here we describe serial capture affinity purification (SCAP), which can be used for the study of protein interactions in live cells and, when combined with cross-linking mass spectrometry, contribute distance restraints for integrative structural modeling. This broadly applicable technology can be used to study any protein complex in human tissue culture cells. We demonstrate SCAP capabilities on a poorly characterized epigenetic protein complex with roles in human cancer.
The INhibitor of Growth (ING) proteins (ING1, ING2, ING3, ING4 and ING5) are a family of epigenetic regulators. Their decreased expression in numerous cancers led to identifying the ING proteins as gatekeeper tumor suppressors as they regulate cell cycle progression, apoptosis and senescence. Subsequently, they were also described as caretaker tumor suppressors through their involvement in DNA replication and the DNA damage response (DDR). Recent studies have identified new interactions of the ING proteins with proteins or pathways implicated in cell proliferation, the maintenance of stem cells pluripotency or the DDR. Furthermore, the ING proteins have been identified as regulators of ribosomal RNA synthesis and of mRNA stability and as regulators of mitochondrial DNA transcription resulting in the regulation of metabolism. These new findings highlight new antitumorigenic activities of the ING proteins that are potential targets for cancer treatment.
Chromatin-modifying complexes containing histone deacetylase (HDAC) activities play critical roles in the regulation of gene transcription in eukaryotes. These complexes are thought to lack intrinsic DNA-binding activity, but according to a well-established paradigm, they are recruited via protein-protein interactions by gene-specific transcription factors and post-translational histone modifications to their sites of action on the genome. The mammalian Sin3L/Rpd3L complex, comprising more than a dozen different polypeptides, is an ancient HDAC complex found in diverse eukaryotes. The subunits of this complex harbor conserved domains and motifs of unknown structure and function. Here we show that Sds3, a constitutively-associated subunit critical for the proper functioning of the Sin3L/Rpd3L complex, harbors a type of Tudor domain that we designate the capped Tudor domain (CTD). Unlike canonical Tudor domains that bind modified histones, the Sds3 CTD binds to nucleic acids that can form higher-order structures such as G-quadruplexes, and shares similarities with the knotted Tudor domain of the Esa1 histone acetyltransferase (HAT) that was previously shown to bind single-stranded RNA. Our findings expand the range of macromolecules capable of recruiting the Sin3L/Rpd3L complex and draw attention to potentially new biological roles for this HDAC complex.
ResearchGate has not been able to resolve any references for this publication.