Approximately 70 human RNA-binding proteins (RBPs) contain a prion-like domain (PrLD). PrLDs are low-complexity domains that possess a similar amino acid composition to prion domains in yeast, which ...enable several proteins, including Sup35 and Rnq1, to form infectious conformers, termed prions. In humans, PrLDs contribute to RBP function and enable RBPs to undergo liquid-liquid phase transitions that underlie the biogenesis of various membraneless organelles. However, this activity appears to render RBPs prone to misfolding and aggregation connected to neurodegenerative disease. Indeed, numerous RBPs with PrLDs, including TDP-43 (transactivation response element DNA-binding protein 43), FUS (fused in sarcoma), TAF15 (TATA-binding protein-associated factor 15), EWSR1 (Ewing sarcoma breakpoint region 1), and heterogeneous nuclear ribonucleoproteins A1 and A2 (hnRNPA1 and hnRNPA2), have now been connected via pathology and genetics to the etiology of several neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal dementia, and multisystem proteinopathy. Here, we review the physiological and pathological roles of the most prominent RBPs with PrLDs. We also highlight the potential of protein disaggregases, including Hsp104, as a therapeutic strategy to combat the aberrant phase transitions of RBPs with PrLDs that likely underpin neurodegeneration.
Structural maintenance of chromosome (SMC) protein complexes are the key organizers of the spatiotemporal structure of chromosomes. The condensin SMC complex has recently been shown to be a molecular ...motor that extrudes large loops of DNA, but the mechanism of this unique motor remains elusive. Using atomic force microscopy, we show that budding yeast condensin exhibits mainly open 'O' shapes and collapsed 'B' shapes, and it cycles dynamically between these two states over time, with ATP binding inducing the O to B transition. Condensin binds DNA via its globular domain and also via the hinge domain. We observe a single condensin complex at the stem of extruded DNA loops, where the neck size of the DNA loop correlates with the width of the condensin complex. The results are indicative of a type of scrunching model in which condensin extrudes DNA by a cyclic switching of its conformation between O and B shapes.
Covalent modification of histones is important in regulating chromatin dynamics and transcription. One example of such modification is ubiquitination, which mainly occurs on histones H2A and H2B. ...Although recent studies have uncovered the enzymes involved in histone H2B ubiquitination and a 'cross-talk' between H2B ubiquitination and histone methylation, the responsible enzymes and the functions of H2A ubiquitination are unknown. Here we report the purification and functional characterization of an E3 ubiquitin ligase complex that is specific for histone H2A. The complex, termed hPRC1L (human Polycomb repressive complex 1-like), is composed of several Polycomb-group proteins including Ring1, Ring2, Bmi1 and HPH2. hPRC1L monoubiquitinates nucleosomal histone H2A at lysine 119. Reducing the expression of Ring2 results in a dramatic decrease in the level of ubiquitinated H2A in HeLa cells. Chromatin immunoprecipitation analysis demonstrated colocalization of dRing with ubiquitinated H2A at the PRE and promoter regions of the Drosophila Ubx gene in wing imaginal discs. Removal of dRing in SL2 tissue culture cells by RNA interference resulted in loss of H2A ubiquitination concomitant with derepression of Ubx. Thus, our studies identify the H2A ubiquitin ligase, and link H2A ubiquitination to Polycomb silencing.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like ...conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the
Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.
Autophagosome formation in yeast entails starvation-induced assembly of the pre-autophagosomal structure (PAS), in which multiple Atg1 complexes (composed of Atg1, Atg13, and the Atg17-Atg29-Atg31 ...subcomplex) are initially engaged. However, the molecular mechanisms underlying the multimeric assembly of these complexes remain unclear. Using structural and biological techniques, we herein demonstrate that Atg13 has a large intrinsically disordered region (IDR) and interacts with two distinct Atg17 molecules using two binding regions in the IDR. We further reveal that these two binding regions are essential not only for Atg1 complex assembly in vitro, but also for PAS organization in vivo. These findings underscore the structural and functional significance of the IDR of Atg13 in autophagy initiation: Atg13 provides intercomplex linkages between Atg17-Atg29-Atg31 complexes, thereby leading to supramolecular self-assembly of Atg1 complexes, in turn accelerating the initial events of autophagy, including autophosphorylation of Atg1, recruitment of Atg9 vesicles, and phosphorylation of Atg9 by Atg1.
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•An essential autophagy factor Atg13 has a large intrinsically disordered region (IDR)•The IDR of Atg13 contains two distinct regions binding intermolecularly to Atg17•Tethering two Atg17 molecules by Atg13 leads to supramolecular Atg1 complex assembly•The Atg13-mediated supramolecular assembly is responsible for autophagy initiation
Yamamoto et al. uncover insights into early steps of yeast autophagosome assembly, where autophagy-related (Atg) proteins localize to a perivacuolar site to organize the pre-autophagosomal structure (PAS). Atg13, via an intrinsically disordered region, provides a flexible linkage between two Atg17 molecules to mediate multimeric Atg1 complex self-assembly for PAS organization.
Orderly termination of sister-chromatid cohesion during mitosis is critical for accurate chromosome segregation. During prophase, mitotic kinases phosphorylate cohesin and its protector sororin, ...triggering Wapl-dependent cohesin release from chromosome arms. The shugoshin (Sgo1)-PP2A complex protects centromeric cohesin until its cleavage by separase at anaphase onset. Here, we report the crystal structure of a human cohesin subcomplex comprising SA2 and Scc1. Multiple HEAT repeats of SA2 form a dragon-shaped structure. Scc1 makes extensive contacts with SA2, with one binding hotspot. Sgo1 and Wapl compete for binding to a conserved site on SA2-Scc1. At this site, mutations of SA2 residues that disrupt Wapl binding bypass the Sgo1 requirement in cohesion protection. Thus, in addition to recruiting PP2A to dephosphorylate cohesin and sororin, Sgo1 physically shields cohesin from Wapl. This unexpected, direct antagonism between Sgo1 and Wapl augments centromeric cohesion protection.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
All BH3-only proteins, key initiators of programmed cell death, interact tightly with multiple binding partners and have sequences of low complexity, properties that are the hallmark of intrinsically ...unstructured proteins (IUPs). We show, using spectroscopic methods, that the BH3-only proteins Bim, Bad and Bmf are unstructured in the absence of binding partners. Detailed sequence analyses are consistent with this observation and suggest that most BH3-only proteins are unstructured. When Bim binds and inactivates prosurvival proteins, most residues remain disordered, only the BH3 element becomes structured, and the short alpha-helical molecular recognition element can be considered to behave as a 'bead on a string'. Coupled folding and binding is typical of many IUPs that have important signaling roles, such as BH3-only proteins, as the inherent structural plasticity favors interaction with multiple targets. This understanding offers promise for the development of BH3 mimetics, as multiple modes of binding are tolerated.
Telomerase-negative cancer cells can maintain their telomeres by a recombination-mediated alternative lengthening of telomeres (ALT) process. We reported previously that sequestration of ...MRE11/RAD50/NBS1 complexes represses ALT-mediated telomere length maintenance, and suppresses formation of ALT-associated promyelocytic leukemia (PML) bodies (APBs). APBs are PML bodies containing telomeric DNA and telomere-binding proteins, and are observed only in a small fraction of cells within asynchronously dividing ALT-positive cell populations. Here, we report that methionine restriction caused a reversible arrest in G0/G1 phase of the cell cycle and reversible induction of APB formation in most cells within an ALT-positive population. We combined methionine restriction with RNA interference to test whether the following proteins are required for APB formation: PML body-associated proteins, PML and Sp100; telomere-associated proteins, TRF1, TRF2, TIN2 and RAP1; and DNA repair proteins, MRE11, RAD50, NBS1 and 53BP1. APB formation was not decreased by depletion of Sp100 (as reported previously) or of 53BP1, although 53BP1 partially colocalizes with APBs. Depletion of the other proteins suppressed APB formation. Because of the close linkage between ALT-mediated telomere maintenance and ability to form APBs, the eight proteins identified by this screen as being required for APB formation are also likely to be required for the ALT mechanism.
Bone marrow stromal cells (BMSCs) and osteoclasts (OCs) confer multiple myeloma (MM) cell survival through elaborating factors. We demonstrate herein that IL-6 and TNF family cytokines, TNFα, BAFF ...and APRIL, but not IGF-1 cooperatively enhance the expression of the serine/threonine kinase Pim-2 in MM cells. BMSCs and OCs upregulate Pim-2 expression in MM cells largely via the IL-6/STAT3 and NF-κB pathway, respectively. Pim-2 short interfering RNA reduces MM cell viability in cocultures with BMSCs or OCs. Thus, upregulation of Pim-2 appears to be a novel anti-apoptotic mechanism for MM cell survival. Interestingly, the mammalian target of rapamycin inhibitor rapamycin further suppresses the MM cell viability in combination with the Pim-2 silencing. The Pim inhibitor (Z)-5-(4-propoxybenzylidene) thiazolidine-2, 4-dione and the PI3K inhibitor LY294002 cooperatively enhance MM cell death. The Pim inhibitor suppresses 4E-BP1 phosphorylation along with the reduction of Mcl-1 and c-Myc. Pim-2 may therefore become a new target for MM treatment.
Mitochondria are essential organelles with numerous functions in cellular metabolism and homeostasis. Most of the >1,000 different mitochondrial proteins are synthesized as precursors in the cytosol ...and are imported into mitochondria by five transport pathways. The protein import machineries of the mitochondrial membranes and aqueous compartments reveal a remarkable variability of mechanisms for protein recognition, translocation, and sorting. The protein translocases do not operate as separate entities but are connected to each other and to machineries with functions in energetics, membrane organization, and quality control. Here, we discuss the versatility and dynamic organization of the mitochondrial protein import machineries. Elucidating the molecular mechanisms of mitochondrial protein translocation is crucial for understanding the integration of protein translocases into a large network that controls organelle biogenesis, function, and dynamics.
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