Notes

Quantitative Characterization of the Volume and also Amount of (One particular,Several)-β-d-glucan with regard to Useful along with Mechanistic Investigation involving Candica (A single,3)-β-d-glucan Synthase.
Peripheral myelin protein 22 (PMP22) folds and trafficks inefficiently, with only 20% of newly expressed protein trafficking to the cell surface. This behavior is exacerbated in many of the mutants associated with Charcot-Marie-Tooth disease, motivating further study. Here we characterized the role of N-glycosylation in limiting PMP22 trafficking. We first eliminated N-glycosylation using an N41Q mutation, which resulted in an almost 3-fold increase in trafficking efficiency of wildtype (WT) PMP22 and a 10-fold increase for the severely unstable L16P disease mutant in HEK293 cells, with similar results in Schwann cells. selleckchem Total cellular levels were also much higher for the WT/N41Q mutant, although not for the L16P/N41Q form. Depletion of oligosaccharyltransferase OST-A and OST-B subunits revealed that WT PMP22 is N-glycosylated posttranslationally by OST-B, whereas L16P is cotranslationally glycosylated by OST-A. Quantitative proteomic screens revealed similarities and differences in the interactome for WT, glycosylation-deficient, and unstable mutant forms of PMP22 and also suggested that L16P is sequestered at earlier stages of endoplasmic reticulum quality control. CRISPR knockout studies revealed a role for retention in endoplasmic reticulum sorting receptor 1 (RER1) in limiting the trafficking of all three forms, for UDP-glucose glycoprotein glucosyltransferase 1 (UGGT1) in limiting the trafficking of WT and L16P but not N41Q, and calnexin (CNX) in limiting the trafficking of WT and N41Q but not L16P. This work shows that N-glycosylation is a limiting factor to forward trafficking PMP22 and sheds light on the proteins involved in its quality control.The Cdk8 kinase module (CKM) is a dissociable part of the coactivator complex mediator, which regulates gene transcription by RNA polymerase II. The CKM has both negative and positive functions in gene transcription that remain poorly understood at the mechanistic level. In order to reconstitute the role of the CKM in transcription initiation, we prepared recombinant CKM from the yeast Saccharomyces cerevisiae. We showed that CKM bound to the core mediator (cMed) complex, sterically inhibiting cMed from binding to the polymerase II preinitiation complex (PIC) in vitro. We further showed that the Cdk8 kinase activity of the CKM weakened CKM-cMed interaction, thereby facilitating dissociation of the CKM and enabling mediator to bind the PIC in order to stimulate transcription initiation. Finally, we report that the kinase activity of Cdk8 is required for gene activation during the stressful condition of heat shock in vivo but not under steady-state growth conditions. Based on these results, we propose a model in which the CKM negatively regulates mediator function at upstream-activating sequences by preventing mediator binding to the PIC at the gene promoter. However, during gene activation in response to stress, the Cdk8 kinase activity of the CKM may release mediator and allow its binding to the PIC, thereby accounting for the positive function of CKM. This may impart improved adaptability to stress by allowing a rapid transcriptional response to environmental changes, and we speculate that a similar mechanism in metazoans may allow the precise timing of developmental transcription programs.Squalene monooxygenase (SM, also known as squalene epoxidase) is a rate-limiting enzyme of cholesterol synthesis that converts squalene to monooxidosqualene and is oncogenic in numerous cancer types. SM is subject to feedback regulation via cholesterol-induced proteasomal degradation, which depends on its lipid-sensing N-terminal regulatory domain. We previously identified an endogenous truncated form of SM with a similar abundance to full-length SM, but whether this truncated form is functional or subject to the same regulatory mechanisms as full-length SM is not known. Here, we show that truncated SM differs from full-length SM in two major ways it is cholesterol resistant and adopts a peripheral rather than integral association with the endoplasmic reticulum membrane. However, truncated SM retains full SM activity and is therefore constitutively active. Truncation of SM occurs during its endoplasmic reticulum-associated degradation and requires the proteasome, which partially degrades the SM N-terminus and disrupts cholesterol-sensing elements within the regulatory domain. Furthermore, truncation relies on a ubiquitin signal that is distinct from that required for cholesterol-induced degradation. Using mutagenesis, we demonstrate that partial proteasomal degradation of SM depends on both an intrinsically disordered region near the truncation site and the stability of the adjacent catalytic domain, which escapes degradation. These findings uncover an additional layer of complexity in the post-translational regulation of cholesterol synthesis and establish SM as the first eukaryotic enzyme found to undergo proteasomal truncation.Proper dendrite morphogenesis and synapse formation are essential for neuronal development and function. Dasm1, a member of the immunoglobulin superfamily, is known to promote dendrite outgrowth and excitatory synapse maturation in vitro. However, the in vivo function of Dasm1 in neuronal development and the underlying mechanisms are not well understood. To learn more, Dasm1 knockout mice were constructed and employed to confirm that Dasm1 regulates dendrite arborization and spine formation in vivo. We performed a yeast two-hybrid screen using Dasm1, revealing MRCKβ as a putative partner; additional lines of evidence confirmed this interaction and identified cytoplasmic proline-rich region (823-947 aa) of Dasm1 and MRCKβ self-activated kinase domain (CC1, 410-744 aa) as necessary and sufficient for binding. Using co-immunoprecipitation assay, autophosphorylation assay, and BS3 cross-linking assay, we show that Dasm1 binding triggers a change in MRCKβ's conformation and subsequent dimerization, resulting in autophosphorylation and activation. Activated MRCKβ in turn phosphorylates a class 2 regulatory myosin light chain, which leads to enhanced actin rearrangement, causing the dendrite outgrowth and spine formation observed before. Removal of Dasm1 in mice leads to behavioral abnormalities. Together, these results reveal a crucial molecular pathway mediating cell surface and intracellular signaling communication to regulate actin dynamics and neuronal development in the mammalian brain.
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