EMBO Reports最新文献

The serine/threonine protein phosphatase 5 (PP5) regulates hormone and stress-induced signaling networks. Unlike other phosphoprotein phosphatases, PP5 contains both regulatory and catalytic domains and is further regulated through post-translational modifications (PTMs). Here we identify that SUMOylation of K430 in the catalytic domain of PP5 regulates phosphatase activity. Additionally, phosphorylation of PP5-T362 is pre-requisite for SUMOylation, suggesting the ordered addition of PTMs regulates PP5 function in cells. Using the glucocorticoid receptor, a well known substrate for PP5, we demonstrate that SUMOylation results in substrate release from PP5. We harness this information to create a non-SUMOylatable K430R mutant as a 'substrate trap' and globally identified novel PP5 substrate candidates. Lastly, we generated a consensus dephosphorylation motif using known substrates, and verified its presence in the new candidate substrates. This study unravels the impact of cross talk of SUMOylation and phosphorylation on PP5 phosphatase activity and substrate release in cells.

Glucose-stimulated insulin secretion (GSIS) from pancreatic beta cells is a principal mechanism for systemic glucose homeostasis, of which regulatory mechanisms are still unclear. Here we show that kinesin molecular motor KIF5B is essential for GSIS through maintaining the voltage-gated calcium channel Ca_V1.2 levels, by facilitating an Hsp70-to-Hsp90 chaperone exchange to pass through the quality control in the endoplasmic reticulum (ER). Phenotypic analyses of KIF5B conditional knockout (cKO) mouse beta cells revealed significant abolishment of glucose-stimulated calcium transients, which altered the behaviors of insulin granules via abnormally stabilized cortical F-actin. KIF5B and Hsp90 colocalize to microdroplets on ER sheets, where Ca_V1.2 but not K_ir6.2 is accumulated. In the absence of KIF5B, Ca_V1.2 fails to be transferred from Hsp70 to Hsp90 via STIP1, and is likely degraded via the proteasomal pathway. KIF5B and Hsc70 overexpression increased Ca_V1.2 expression via enhancing its chaperone binding. Thus, ER sheets may serve as the place of KIF5B- and Hsp90-dependent chaperone exchange, which predominantly facilitates Ca_V1.2 production in beta cells and properly enterprises GSIS against diabetes.

Apidaecin 1b (Api), the first characterized Type II Proline-rich antimicrobial peptide (PrAMP), is encoded in the honey bee genome. It inhibits bacterial growth by binding in the nascent peptide exit tunnel of the ribosome after the release of the completed protein and trapping the release factors. By genome mining, we have identified 71 PrAMPs encoded in insect genomes as pre-pro-polyproteins. Having chemically synthesized and tested the activity of 26 peptides, we demonstrate that despite significant sequence variation in the N-terminal sequence, the majority of the PrAMPs that retain the conserved C-terminal sequence of Api are able to trap the ribosome at the stop codons and induce stop codon readthrough-all hallmarks of Type II PrAMP mode of action. Some of the characterized PrAMPs exhibit superior antibacterial activity in comparison with Api. The newly solved crystallographic structures of the ribosome complexed with Api and with the more active peptide Fva1 from the stingless bee demonstrate the universal placement of the PrAMPs' C-terminal pharmacophore in the post-release ribosome despite variations in their N-terminal sequence.

The nucleoskeleton is essential for nuclear architecture as well as genome integrity and gene expression. In addition to lamins, titin or spectrins, dynamic actin filament polymerization has emerged as a potential intranuclear structural element but its functions are less well explored. Here we found that calcium elevations trigger rapid nuclear actin assembly requiring the nuclear membrane protein SUN2 independently of its function as a component of the LINC complex. Instead, SUN2 colocalized and associated with the formin and actin nucleator INF2 in the nuclear envelope in a calcium-regulated manner. Moreover, SUN2 is required for active RNA polymerase II (RNA Pol II) clustering in response to calcium elevations. Thus, our data uncover a SUN2-formin module linking the nuclear envelope to intranuclear actin assembly to promote signal-dependent spatial reorganization of active RNA Pol II.

Becker muscular dystrophy (BMD) is an X-linked disorder due to in-frame mutations in the DMD gene, leading to a less abundant and truncated dystrophin. BMD is less common and severe than Duchenne muscular dystrophy (DMD) as well as less investigated. To accelerate the search for innovative treatments, we developed a rat model of BMD by deleting the exons 45-47 of the Dmd gene. Here, we report a functional and histopathological evaluation of these rats during their first year of life, compared to DMD and control littermates. BMD rats exhibit moderate damage to locomotor and diaphragmatic muscles but suffer from a progressive cardiomyopathy. Single nuclei RNA-seq analysis of cardiac samples revealed shared transcriptomic abnormalities in BMD and DMD rats and highlighted an altered end-addressing of TMEM65 and Connexin-43 at the intercalated disc, along with electrocardiographic abnormalities. Our study documents the natural history of a translational preclinical model of BMD and reports a cellular mechanism for the cardiac dysfunction in BMD and DMD offering opportunities to further investigate the organization role of dystrophin in intercellular communication.

In Huntington's disease (HD), aberrant processing of huntingtin (HTT) mRNA produces HTT1a transcripts that encode the pathogenic HTT exon 1 protein. The mechanisms behind HTT1a production are not fully understood. Considering the role of m⁶A in RNA processing and splicing, we investigated its involvement in HTT1a generation. Here, we show that m⁶A methylation is increased before the cryptic poly(A) sites (IpA1 and IpA2) within the huntingtin RNA in the striatum of Hdh+/Q111 mice and human HD samples. We further assessed m⁶A's role in mutant Htt mRNA processing by pharmacological inhibition and knockdown of METTL3, as well as targeted demethylation of Htt intron 1 using a dCas13-ALKBH5 system in HD mouse cells. Our data reveal that Htt1a transcript levels are regulated by both METTL3 and the methylation status of Htt intron 1. They also show that m⁶A methylation in intron 1 depends on expanded CAG repeats. Our findings highlight a potential role for m⁶A in aberrant splicing of Htt mRNA.

Inflammatory bowel disease (IBD) is a disorder causing chronic inflammation in the gastrointestinal tract, and its pathophysiological mechanisms are still under investigation. Here, we find that mice deficient of YOD1, a deubiquitinating enzyme, are highly susceptible to dextran sulfate sodium (DSS)-induced colitis. The bone marrow transplantation experiment reveals that YOD1 derived from hematopoietic cells inhibits DSS colitis. Moreover, YOD1 exerts its protective role by promoting nucleotide-binding oligomerization domain 2 (NOD2)-mediated physiological inflammation in macrophages. Mechanistically, YOD1 inhibits the proteasomal degradation of receptor-interacting serine/threonine kinase 2 (RIPK2) by reducing its K48 polyubiquitination, thereby increasing RIPK2 abundance to enhance NOD2 signaling. Consistently, the protective function of muramyldipeptide, a NOD2 ligand, in experimental colitis is abolished in mice deficient of YOD1. Importantly, YOD1 is upregulated in colon-infiltrating macrophages in patients with colitis. Collectively, this study identifies YOD1 as a novel regulator of colitis.