Short linear motifs (SLiMs) are protein binding modules that play major roles in almost all cellular processes. SLiMs are short, often highly degenerate, difficult to characterize and hard to detect. The eukaryotic linear motif (ELM) resource (elm.eu.org) is dedicated to SLiMs, consisting of a manually curated database of over 275 motif classes and over 3000 motif instances, and a pipeline to discover candidate SLiMs in protein sequences. For 15 years, ELM has been one of the major resources for motif research. In this database update, we present the latest additions to the database including 32 new motif classes, and new features including Uniprot and Reactome integration. Finally, to help provide cellular context, we present some biological insights about SLiMs in the cell cycle, as targets for bacterial pathogenicity and their functionality in the human kinome.
Recent reports suggest that human genogroup II genotype 17 (GII.17) noroviruses are increasing in prevalence. We analyzed the evolutionary changes of three GII.17 capsid protruding (P) domains. We found that the GII.17 P domains had little cross-reactivity with antisera raised against the dominant GII.4 strains. X-ray structural analysis of GII.17 P domains from 2002 to 2014 and 2015 suggested that surface-exposed substitutions on the uppermost part of the P domain might have generated a novel 2014-2015 GII.17 variant. Human noroviruses are the dominant cause of outbreaks of acute gastroenteritis. In the past decade, genogroup II genotype 4 (GII.4) norovirus strains were those mostly responsible for epidemic outbreaks (1-3). However, a GII.17 variant norovirus was found recently to cause an alarming number of outbreaks in certain parts of Asia in 2014 to 2015 (4-8). Before this time, the GII.17 norovirus was only a minor cause of infections, although it was first described in 1978 (9). Researchers are now reporting that the GII.17 variant is emerging in other parts of the world, and molecular epidemiologists have warned that the GII.17 norovirus might replace the predominant GII.4 norovirus (10).Noroviruses have a single-stranded, positive-sense RNA genome of 7.5 to 7.7 kb. The genome contains three open reading frames (ORFs). The first ORF (ORF1) encodes nonstructural proteins, including the RNA-dependent RNA polymerase (RdRp), ORF2 encodes capsid protein (VP1), and ORF3 encodes a minor capsid protein (VP2) (11). The X-ray crystal structure of the prototype (GI.1) virus-like particles (VLPs) identified two domains, the shell (S) domain and the protruding (P) domain, which can be further subdivided into P1 and P2 subdomains (12). The S domain surrounds the viral RNA, whereas the P domain contains the determinants for cell attachment and antigenicity. Human noroviruses are known to bind histo-blood group antigens (HBGAs), and the interaction is thought to be important for infection (13)(14)(15)(16). Two recent reports indicated that, similarly to other GII noroviruses, the recent GII.17 strains bind a panel of different HBGA types (4,8).Human noroviruses are believed to evolve in a manner similar to that seen with influenza viruses, where new norovirus genotype variants emerge every other year. Evolving strains with an ϳ5% amino acid change can reinfect the same individual (17). Data on short-and long-term immunity to human norovirus are still unclear, although vaccines are currently been tested in clinical trials (18,19). Unfortunately, the vaccines, which can include VLPs or P domains (20, 21), may not protect from antigenically divergent strains (18-21). Here, we report the first X-ray crystal structure of GII.17 norovirus P domains and describe the cross-reactivities with antibodies (Abs) raised against GII.4 strains, which are targeted by the current vaccines in clinical trials.Three different GII.17 norovirus strains were selected for antibody binding and structural analysis: a nonprevalent 2002 strain (Sai...
We introduce a family of bright, rhodamine-based calcium indicators with tuneable affinities and colors. The indicators can be specifically localized to different cellular compartments and are compatible with both fluorescence and bioluminescence readouts through conjugation to HaloTag fusion proteins. Importantly, their increase in fluorescence upon localization enables no-wash live-cell imaging, which greatly facilitates their use in biological assays. Applications as fluorescent indicators in rat hippocampal neurons include the detection of single action potentials and of calcium fluxes in the endoplasmic reticulum. Applications as bioluminescent indicators include the recording of the pharmacological modulation of nuclear calcium in high-throughput compatible assays. The versatility and remarkable ease of use of these indicators make them powerful tools for bioimaging and bioassays.
Mutations in the tumor suppressor p53 are among the most highly occurring events in colorectal cancer (CRC). Such mutations have been shown to influence the sensitivity of cancer cells to chemotherapeutic agents. However their impact on the efficacy of the proteasomal inhibitor bortezomib remains controversial. We thus re-evaluated the toxicity of bortezomib in the CRC cell lines HCT116 wt (wild-type) and its p53−/− clone. Transient resistance to bortezomib treatment was observed in p53-null cells that was later accompanied by an increase in levels and nuclear translocation of TAp73, an isoform of the p53-homologue p73, as well as induction of apoptosis. Knockdown of p73 in p53−/− cells using CRISPR/Cas9 significantly prolonged the duration of resistance. Moreover, similar results were observed in HT-29 cells carrying mutated p53, but not human fibroblasts with expression of functional p53. Thus, our results clearly demonstrated that TAp73 served as a substitute for p53 in bortezomib-induced apoptosis in p53-deficient or mutated cells, implicating that TAp73 could be a potential therapeutic target for treatment of CRCs, in particular those lacking functional p53.
Neurons are highly asymmetric cells that span long distances and need to react promptly to local demands. Consequently, neuronal secretory pathway elements are distributed throughout neurites, specifically in post-synaptic compartments, to enable local protein synthesis and delivery. Whether and how changes in local synaptic activity correlate to post-synaptic secretory elements is still unclear. To assess this, we used STED nanoscopy and automated quantitative image analysis of post-synaptic markers of the endoplasmic reticulum, ER-Golgi intermediate compartment, trans-Golgi network, and spine apparatus. We found that the distribution of these proteins was dependent on pre-synaptic activity, measured as the amount of recycling vesicles. Moreover, their abundance correlated to both pre- and post-synaptic markers of synaptic strength. Overall, the results suggest that in small, low-activity synapses the secretory pathway components are tightly clustered in the synaptic area, presumably to enable rapid local responses, while bigger synapses utilise secretory machinery components from larger, more diffuse areas.
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