FEBS Letters最新文献

Formation of clathrin-coated vesicles (CCVs) in receptor-mediated endocytosis is a mechanistically well-established process, in which clathrin, the adaptor protein complex AP-2, and the large GTPase dynamin play crucial roles. In order to obtain more mechanistic insight into this process, here we established a giant unilamellar vesicle (GUV)-based in vitro CCV reconstitution system with chemically defined components and the full-length recombinant proteins clathrin, AP-2, epsin-1, and dynamin-2. Our results support the predominant model in which hydrolysis of GTP by dynamin is a prerequisite to generate CCVs. Strikingly, in this system at near physiological concentrations of reagents, epsin-1 alone does not have the propensity for scission but is required for bud formation, whereas AP-2 and clathrin are not sufficient. Thus, our study reveals that epsin-1 is an important factor for the maturation of clathrin coated buds, a prerequisite for vesicle generation.

The sorting of RNAs to specific regions of the cell for local translation represents an important mechanism directing protein distribution and cell compartmentalization. While significant progress has been made in understanding the mechanisms underlying the transport and localization of mRNAs, the mechanisms governing ribosome mobilization are less well understood. Ribosomes present in the cytoplasm of multiple cell types can form ribonucleoprotein complexes that also contain myosin-Va (Myo5a), a processive, actin-dependent molecular motor. Here, we report that Myo5a can be disassociated from ribosomes when ribonucleoprotein complexes are exposed to calcium, both in vitro and in vivo. We suggest that Myo5a may act as a molecular switch able to anchor or release ribosomes from the actin cytoskeleton in response to intracellular signaling.

Glycoside hydrolases catalyze the hydrolysis of glycosidic linkages in carbohydrates. The glycoside hydrolase family 31 (GH31) contains α-glucosidase, α-xylosidase, α-galactosidase, and α-transglycosylase. Recent work has expanded the diversity of substrate specificity of GH31 enzymes, and α-N-acetylgalactosaminidases (αGalNAcases) belonging to GH31 have been identified in human gut bacteria. Here, we determined the first crystal structure of a truncated form of GH31 αGalNAcase from the human gut bacterium Enterococcus faecalis. The enzyme has a similar fold to other reported GH31 enzymes and an additional fibronectin type 3-like domain. Additionally, the structure in complex with N-acetylgalactosamine reveals that conformations of the active site residues, including its catalytic nucleophile, change to recognize the ligand. Our structural analysis provides insight into the substrate recognition and catalytic mechanism of GH31 αGalNAcases.

The Sin3L/Rpd3L histone deacetylase (HDAC) complex is one of six major HDAC complexes in the nucleus, and its recruitment by promoter-bound transcription factors is an important step in many gene transcription regulatory pathways. Here, we investigate how the Myt1L zinc finger transcription factor, important for neuronal differentiation and the maintenance of neuronal identity, recruits this complex at the molecular level. We show that Myt1L, through a highly conserved segment shared with its paralogs, interacts directly and specifically with the Sin3 PAH1 domain, binding principally to the canonical hydrophobic cleft found in paired amphipathic helix domain (PAH) domains. Our findings are relevant not only for other members of the Myt family but also for enhancing our understanding of the rules of protein-protein interactions involving Sin3 PAH domains.

RecJ homologs, which occur in virtually all prokaryotes and eukaryotes, play key roles in DNA damage repair and recombination. Current evidence shows that RecJ family proteins exhibit exonuclease activity, degrading single-stranded nucleic acids. Here, we report a novel RecJ isolated from Bacillus halodurans, which utilizes double-stranded DNA as a substrate and functions as an endonuclease. Bacillus halodurans RecJ (BhRecJ) cleaves supercoiled plasmids into open circular and linear forms. Besides the typical domains of DHH, DHHA1, and oligonucleotide-binding-fold, BhRecJ possesses a C-terminal domain with unknown function, which might form the core of the endonuclease activity. Using mutational analysis, we mapped several essential residues for BhRecJ endonuclease activity. Our findings suggest that BhRecJ may be involved in biological processes not typically associated with RecJ proteins.

Receptor-interacting serine/threonine-protein kinase 3 (RIPK3) is a central protein in necroptosis with great potential as a target for treating necroptosis-associated diseases, such as Crohn's disease. However, blockade of RIPK3 kinase activity leads to unexpected RIPK3-initiated apoptosis. Herein, we found that PP2, a known SRC inhibitor, inhibits TNF-α-induced necroptosis without initiating apoptosis. Further investigation showed that PP2 acts as an inhibitor of not only SRC but also RIPK3. PP2 does not disturb the integrity of the RIPK1-RIPK3-mixed lineage kinase domain-like pseudokinase (MLKL) necroptosome or the autophosphorylation of RIPK3 at T231/S232 but disrupts RIPK3 oligomerization, thereby impairing the phosphorylation and oligomerization of MLKL. These results demonstrate the essential role of RIPK3 oligomerization in necroptosis and suggest a potential RIPK3 oligomerization-targeting strategy for therapeutic development.

The signalling pathway governing general control nonderepressible (Gcn)2 kinase allows cells to cope with amino acid shortage. Under starvation, Gcn2 phosphorylates the translation initiation factor eukaryotic translation initiation factor (eIF)2α, triggering downstream events that ultimately allow cells to cope with starvation. Under nutrient-replete conditions, the translation elongation factor eEF1A binds Gcn2 to contribute to keeping Gcn2 inactive. Here, we aimed to map the regions in eEF1A involved in binding and/or regulating Gcn2. We find that eEF1A amino acids 1-221 and 222-315, containing most of domains I and II, respectively, bind Gcn2 in vitro. Overexpression of eEF1A lacking or containing domain III impairs eIF2α phosphorylation. While the latter reduces growth under starvation similarly to eEF1A lacking domain I, the former enhances growth in a Gcn2-dependent manner. Our studies suggest that domain II is required for Gcn2 inhibition and that eEF1A lacking domain III mainly affects the Gcn2 response pathway downstream of Gcn2.

Membrane traffic between secretory and endosomal compartments is vesicle-mediated and must be tightly balanced to maintain a physiological compartment size. Vesicle formation is initiated by guanine nucleotide exchange factors (GEFs) that activate the ARF family of small GTPases. Regulatory mechanisms, including reversible phosphorylation, allow ARF-GEFs to support vesicle formation only at the right time and place in response to cellular needs. Here, we review current knowledge of how the Golgi-specific brefeldin A-resistance factor 1 (GBF1)/brefeldin A-inhibited guanine nucleotide exchange protein (BIG) family of ARF-GEFs is influenced by phosphorylation and use predictive paradigms to propose new regulatory paradigms. We describe a conserved cluster of phosphorylation sites within the N-terminal domains of the GBF1/BIG ARF-GEFs and suggest that these sites may respond to homeostatic signals related to cell growth and division. In the C-terminal region, GBF1 shows phosphorylation sites clustered differently as compared with the similar configuration found in both BIG1 and BIG2. Despite this similarity, BIG1 and BIG2 phosphorylation patterns are divergent in other domains. The different clustering of phosphorylation sites suggests that the nonconserved sites may represent distinct regulatory nodes and specify the function of GBF1, BIG1, and BIG2.

TDP-43 is the major pathogenic protein of amyotrophic lateral sclerosis (ALS). Previously, we identified that TDP-43 interacts with G-quadruplex (G4)-containing RNA and is involved in their long-distance transport in neurons. For the molecular dissection of the TDP-43 and G4-RNA interaction, we analyzed it here in vitro and in cultured cells using a set of 10 mutant TDP-43 proteins from familial and sporadic ALS patients as well as using the TDP-43 C-terminal Gly-rich domain alone. Our results altogether indicate the involvement of the Gly-rich region of TDP-43 in the initial recognition and binding of G4-RNA, which then induces tight binding of TDP-43 with target RNAs, supposedly in conjunction with its RNA recognition motifs.