Cornelia de Lange syndrome (CdLS) is a dominantly inherited congenital malformation disorder, caused by mutations in the cohesin-loading protein NIPBL for nearly 60% of individuals with classical ...CdLS, and by mutations in the core cohesin components SMC1A (~5%) and SMC3 (<1%) for a smaller fraction of probands. In humans, the multisubunit complex cohesin is made up of SMC1, SMC3, RAD21 and a STAG protein. These form a ring structure that is proposed to encircle sister chromatids to mediate sister chromatid cohesion and also has key roles in gene regulation. SMC3 is acetylated during S-phase to establish cohesiveness of chromatin-loaded cohesin, and in yeast, the class I histone deacetylase Hos1 deacetylates SMC3 during anaphase. Here we identify HDAC8 as the vertebrate SMC3 deacetylase, as well as loss-of-function HDAC8 mutations in six CdLS probands. Loss of HDAC8 activity results in increased SMC3 acetylation and inefficient dissolution of the ‘used’ cohesin complex released from chromatin in both prophase and anaphase. SMC3 with retained acetylation is loaded onto chromatin, and chromatin immunoprecipitation sequencing analysis demonstrates decreased occupancy of cohesin localization sites that results in a consistent pattern of altered transcription seen in CdLS cell lines with either NIPBL or HDAC8 mutations.
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DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Inactivation of the transcription factor p53, through either direct mutation or aberrations in one of its many regulatory pathways, is a hallmark of virtually every tumor. In recent years, screening ...for p53 activators and a better understanding of the molecular mechanisms of oncogenic perturbations of p53 function have opened up a host of novel avenues for therapeutic intervention in cancer: from the structure-guided design of chemical chaperones to restore the function of conformationally unstable p53 cancer mutants, to the development of potent antagonists of the negative regulators MDM2 and MDMX and other modulators of the p53 pathway for the treatment of cancers with wild-type p53. Some of these compounds have now moved from proof-of-concept studies into clinical trials, with prospects for further, personalized anticancer medicines. We trace the structural evolution of the p53 pathway, from germ-line surveillance in simple multicellular organisms to its pluripotential role in humans.
Apoptosis is initiated when Bcl-2 and its prosurvival relatives are engaged by proapoptotic BH3-only proteins via interaction of its BH3 domain with a groove on the Bcl-2-like proteins. These ...interactions have been considered promiscuous, but our analysis of the affinity of eight BH3 peptides for five Bcl-2-like proteins has revealed that the interactions vary over 10,000-fold in affinity, and accordingly, only certain protein pairs associate inside cells. Bim and Puma potently engaged all the prosurvival proteins comparably. Bad, however, bound tightly to Bcl-2, Bcl-x
L, and Bcl-w but only weakly to A1 and not to Mcl-1. Strikingly, Noxa bound only Mcl-1 and A1. In accord with their complementary binding, Bad and Noxa cooperated to induce potent killing. The results suggest that apoptosis relies on selective interactions between particular subsets of these proteins and that it should be feasible to discover BH3-mimetic drugs that inactivate specific prosurvival targets.
The microtubule-associated protein tau forms insoluble, amyloid-type aggregates in various dementias, most notably Alzheimer's disease. Cellular chaperone proteins play important roles in maintaining ...protein solubility and preventing aggregation in the crowded cellular environment. Although tau is known to interact with numerous chaperones, it remains unclear how these chaperones function mechanistically to prevent tau aggregation and how chaperones from different classes compare in terms of mechanism. Here, we focused on the small heat shock protein HspB1 (also known as Hsp27) and the constitutive chaperone Hsc70 (also known as HspA8) and report how each chaperone interacts with tau to prevent its fibril formation. Using fluorescence and NMR spectroscopy, we show that the two chaperones inhibit tau fibril formation by distinct mechanisms. HspB1 delayed tau fibril formation by weakly interacting with early species in the aggregation process, whereas Hsc70 was highly efficient at preventing tau fibril elongation, possibly by capping the ends of tau fibrils. Both chaperones recognized aggregation-prone motifs within the microtubule-binding repeat region of tau. However, HspB1 binding remained transient in both aggregation-promoting and non-aggregating conditions, whereas Hsc70 binding was significantly tighter under aggregation-promoting conditions. These differences highlight the fact that chaperones from different families play distinct but complementary roles in the prevention of pathological protein aggregation.
Methylation of histones has been regarded as a stable modification defining the epigenetic program of the cell, which regulates chromatin structure and transcription. However, the recent discovery of ...histone demethylases has challenged the stable nature of histone methylation. Here we demonstrate that the JARID1 proteins RBP2, PLU1, and SMCX are histone demethylases specific for di- and trimethylated histone 3 lysine 4 (H3K4). Consistent with a role for the JARID1 Drosophila homolog Lid in regulating expression of homeotic genes during development, we show that RBP2 is displaced from Hox genes during embryonic stem (ES) cell differentiation correlating with an increase of their H3K4me3 levels and expression. Furthermore, we show that mutation or RNAi depletion of the C. elegans JARID1 homolog rbr-2 leads to increased levels of H3K4me3 during larval development and defects in vulva formation. Taken together, these results suggest that H3K4me3/me2 demethylation regulated by the JARID1 family plays an important role during development.
Precise control of mesenchymal stem cell (MSC) differentiation is critical for tissue development and regeneration. We show here that kindlin-2 is a key determinant of MSC fate decision. Depletion of ...kindlin-2 in MSCs is sufficient to induce adipogenesis and inhibit osteogenesis in vitro and in vivo. Mechanistically, kindlin-2 regulates MSC differentiation through controlling YAP1/TAZ at both the transcript and protein levels. Kindlin-2 physically associates with myosin light-chain kinase in response to mechanical cues of cell microenvironment and intracellular signaling events and promotes myosin light-chain phosphorylation. Loss of kindlin-2 inhibits RhoA activation and reduces myosin light-chain phosphorylation, stress fiber formation, and focal adhesion assembly, resulting in increased Ser127 phosphorylation, nuclear exclusion, and ubiquitin ligase atrophin-1 interacting protein 4-mediated degradation of YAP1/TAZ. Our findings reveal a novel kindlin-2 signaling axis that senses the mechanical cues of cell microenvironment and controls MSC fate decision, and they suggest a new strategy to regulate MSC differentiation, tissue repair, and regeneration.
Caspase-8 activation can be triggered by death receptor-mediated formation of the death-inducing signaling complex (DISC) and by the inflammasome adaptor ASC. Caspase-8 assembles with FADD at the ...DISC and with ASC at the inflammasome through its tandem death effector domain (tDED), which is regulated by the tDED-containing cellular inhibitor cFLIP and the viral inhibitor MC159. Here we present the caspase-8 tDED filament structure determined by cryoelectron microscopy. Extensive assembly interfaces not predicted by the previously proposed linear DED chain model were uncovered, and were further confirmed by structure-based mutagenesis in filament formation in vitro and Fas-induced apoptosis and ASC-mediated caspase-8 recruitment in cells. Structurally, the two DEDs in caspase-8 use quasi-equivalent contacts to enable assembly. Using the tDED filament structure as a template, structural analyses reveal the interaction surfaces between FADD and caspase-8 and the distinct mechanisms of regulation by cFLIP and MC159 through comingling and capping, respectively.
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•Caspase-8 tDED assembles into filaments through quasi-equivalent contacts•The assembly of caspase-8 filaments is nucleated by the upstream Fas/FADD complex•cFLIP tDED also forms filaments, which interact with caspase-8 by comingling•MC159 inhibits caspase-8 filament assembly by a unique capping mechanism
How caspase-8 is activated has been a long-standing question. Fu et al. show that its tDED forms filaments using quasi-equivalent interactions. Cryo-EM structure of the filament reveals mechanisms of caspase-8 activation and its regulation by cFLIP and MC159.
Mitophagy, the selective removal of damaged or excess mitochondria by autophagy, is an important process in cellular homeostasis. The outer mitochondrial membrane (OMM) proteins NIX, BNIP3, FUNDC1, ...and Bcl2‐L13 recruit ATG8 proteins (LC3/GABARAP) to mitochondria during mitophagy. FKBP8 (also known as FKBP38), a unique member of the FK506‐binding protein (FKBP) family, is similarly anchored in the OMM and acts as a multifunctional adaptor with anti‐apoptotic activity. In a yeast two‐hybrid screen, we identified FKBP8 as an ATG8‐interacting protein. Here, we map an N‐terminal LC3‐interacting region (LIR) motif in FKBP8 that binds strongly to LC3A both in vitro and in vivo. FKBP8 efficiently recruits lipidated LC3A to damaged mitochondria in a LIR‐dependent manner. The mitophagy receptors BNIP3 and NIX in contrast are unable to mediate an efficient recruitment of LC3A even after mitochondrial damage. Co‐expression of FKBP8 with LC3A profoundly induces Parkin‐independent mitophagy. Strikingly, even when acting as a mitophagy receptor, FKBP8 avoids degradation by escaping from mitochondria. In summary, this study identifies novel roles for FKBP8 and LC3A, which act together to induce mitophagy.
Synopsis
FKBP8 (also called FKBP38) is a novel mitophagy receptor that selectively recruits LC3A to damaged mitochondria to mediate Parkin‐independent degradation of mitochondria by autophagy. The anti‐apoptotic FKBP8 itself escapes degradation to ensure cell survival.
FKBP8 binds to LC3A via an N‐terminal LIR motif.
FKBP8 recruits lipidated LC3A to damaged mitochondria in a Parkin‐independent manner.
Co‐expression of FKBP8 and LC3A induces efficient Parkin‐independent mitophagy.
FKBP8 escapes from the mitochondria destined for autophagic degradation after having recruited LC3A.
FKBP8 (also called FKBP38) is a novel mitophagy receptor that selectively recruits LC3A to damaged mitochondria to mediate Parkin‐independent degradation of mitochondria by autophagy. The anti‐apoptotic FKBP8 itself escapes degradation to ensure cell survival.
Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the ...discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α
β
integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.
In two proof‐of‐concept studies, we established and validated the Bacterial Growth Inhibition Screen (BGIS), which explores recombinant protein toxicity in Escherichia coli as a largely overlooked ...and alternative means for basic characterization of functional eukaryotic protein domains. By applying BGIS, we identified an unrecognized RNA‐interacting domain in the DEK oncoprotein (this study) and successfully combined BGIS with random mutagenesis as a screening tool for loss‐of‐function mutants of the DNA modulating domain of DEK 1. Collectively, our findings shed new light on the phenomenon of recombinant protein toxicity in E. coli. Given the easy and rapid implementation and wide applicability, BGIS will extend the repertoire of basic methods for the identification, analysis and unbiased manipulation of proteins.
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