Molecular and Cellular Biology

CCN2/connective tissue growth factor (CTGF) potentially serves as a therapeutic target for chronic kidney disease. Here we investigated CCN2 module-4, encoded by Ccn2 exon 5, through the generation of Ccn2 exon 5 knockout mice ( Ex5-/- mice). To investigate renal fibrosis pathogenesis, Ex5-/- mice were employed to model unilateral ureteral obstruction (UUO), unilateral ischemic-reperfusion injury (UIRI), and 5/6 nephrectomy. Interstitial fibrosis was significantly attenuated in the Ex5-/- mice in the three models...

The circadian clock is driven by a transcriptional-translational feedback loop, and cryptochrome 2 (CRY2) represses CLOCK/BMAL1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the CRY2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of CRY2 that mediates interaction with Period 2 (PER2). We further demonstrate that this mechanism is required for repressing circadian clock-controlled Wnt signaling to promote adipogenesis...

IRES mediated translation initiation requires a different repertoire of factors than canonical cap-dependent translation. Treatments that inhibit the canonical translation factor EIF4G1 have little or no effect on the ability of the Insr and Igf1r cellular IRESes to promote translation. Transcripts for two cellular receptors contain RNA elements that facilitate translation initiation without intact EIF4G1. Cellular IRES mechanisms may resemble viral type III IRESes allowing them to promote translate with a limited number of initiation factors allowing them to work under stress conditions when canonical translation is repressed...

The primary cilium functions as a cellular sensory organelle and signaling antenna that detects and transduces extracellular signals. Mutations in the human gene CILK1 (ciliogenesis associated kinase 1) cause abnormal cilia elongation and faulty Hedgehog signaling, associated with developmental disorders and epilepsy. CILK1 is a protein kinase that requires dual phosphorylation of its TDY motif for activation and its extended C-terminal intrinsically disordered region (IDR) mediates targeting to the basal body and substrate recognition...

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Positive transcription elongation factor b (P-TEFb) regulates expression of diverse sets of genes within mammalian cells that have implications in several human disease pathogeneses. However, mechanisms of functional regulation of P-TEFb complex through regulation of its stability are poorly known. In this study, we show an important role of C-terminus of Hsc70-interacting protein (CHIP aka STUB1) in regulation of overall level of CDK9 and thus P-TEFb complex within mammalian cells. STUB1 acts as a ubiquitin E3 ligase for proteasomal degradation of CDK9 involving N-terminal lysine 3 (K3) residue...

The master tumor suppressor p53 regulates multiple cell fate decisions, such as cell cycle arrest and apoptosis, via transcriptional control of a broad gene network. Dysfunction in the p53 network is common in cancer, often through mutations that inactivate p53 or other members of the pathway. Induction of tumor-specific cell death by restoration of p53 activity without off-target effects has gained significant interest in the field. In this study, we explore the gene regulatory mechanisms underlying a putative anticancer strategy involving stimulation of the p53-independent integrated stress response (ISR)...

Replication fork arrest-induced DNA double strand breaks (DSBs) caused by lesions are effectively suppressed in cells due to the presence of a specialized mechanism, commonly referred to as DNA damage tolerance (DDT). In eukaryotic cells, DDT is facilitated through translesion DNA synthesis (TLS) carried out by a set of DNA polymerases known as TLS polymerases. Another parallel mechanism, referred to as homology-directed DDT, is error-free and involves either template switching or fork reversal. The significance of the DDT pathway is well established...

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for in vitro experimentation. In vitro and in vivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 ( KLF4 ), oe- KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH...

The highly conserved retromer complex controls the fate of hundreds of receptors that pass through the endolysosomal system and is a central regulatory node for diverse metabolic programs. More than 20 years ago, retromer was discovered as an essential regulator of endosome-to-Golgi transport in yeast; since then, significant progress has been made to characterize how metazoan retromer components assemble to enable its engagement with endosomal membranes, where it sorts cargo receptors from endosomes to the trans -Golgi network or plasma membrane through recognition of sorting motifs in their cytoplasmic tails...

Previous research has shown that Δ40p53, the translational isoform of p53, can inhibit cell growth independently of p53 by regulating microRNAs. Here, we explored the role of Δ40p53 in regulating the long noncoding RNA-micro-RNA-cellular process axis, specifically focusing on LINC00176 . Interestingly, LINC00176 levels were predominantly affected by the overexpression/stress-mediated induction and knockdown of Δ40p53 rather than p53 levels. Additional assays revealed that Δ40p53 transactivates LINC00176 transcriptionally and could also regulate its stability...

The complex intrinsic and extrinsic pathways contributing to platelet activation profoundly impact hemostasis and thrombosis. Detailed cellular mechanisms that regulate calcium mobilization, Akt activation, and integrin signaling in platelets remain incompletely understood. Dematin is a broadly expressed actin binding and bundling cytoskeletal adaptor protein regulated by phosphorylation via cAMP-dependent protein kinase. Here, we report the development of a conditional mouse model specifically lacking dematin in platelets...

Activity of the myogenic regulatory protein myocyte enhancer factor-2 (MEF2) is modulated by post-translational modification. We investigated the in vivo phosphorylation of Drosophila MEF2, and identified serine 98 (S98) as a phosphorylated residue. Phospho-mimetic (S98E) and phospho-null (S98A) isoforms of MEF2 did not differ from wild-type in their activity in vitro, so we used CRISPR/Cas9 to generate an S98A allele of the endogenous gene. In mutant larvae we observed phenotypes characteristic of reduced MEF2 function, including reduced body wall muscle size and reduced expression of myofibrillar protein genes; conversely, S98A homozygotes showed enhanced MEF2 function through muscle differentiation within the adult myoblasts associated with the wing imaginal disc...

Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast...

Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is known to associated with fibrosis through its catalytic activity to produce prolyl-tRNA. Although its catalytic inhibitor halofuginone (HF) has been known to inhibit the TGF-β pathway as well as to reduce prolyl-tRNA production for the control of fibrosis, the underlying mechanism how EPRS1 regulates the TGF-β pathway was not fully understood. Here, we show a noncatalytic function of EPRS1 in controlling the TGF-β pathway and hepatic stellate cell activation via its interaction with TGF-β receptor I (TβRI)...

Rif1 mediates telomere length, DNA replication, and DNA damage responses in budding yeast. Previous work identified several posttranslational modifications of Rif1, however none of these was shown to mediate the molecular or cellular responses to DNA damage, including telomere damage. We searched for such modifications using immunoblotting methods and the cdc13-1 and tlc1Δ models of telomere damage. We found that Rif1 is phosphorylated during telomere damage, and that serines 57 and 110 within a novel phospho-gate domain (PGD) of Rif1 are important for this modification, in cdc13-1 cells...

Rho GTPases are global regulators of cell polarity and signaling. By exploring the turnover regulation of the yeast Rho GTPase Cdc42p, we identified new regulatory features surrounding the stability of the protein. We specifically show that Cdc42p is degraded at 37 °C by chaperones through lysine residues located in the C-terminus of the protein. Cdc42p turnover at 37 °C occurred by the 26S proteasome in an ESCRT-dependent manner in the lysosome/vacuole. By analyzing versions of Cdc42p that were defective for turnover, we show that turnover at 37 °C promoted cell polarity but was defective for sensitivity to mating pheromone, presumably mediated through a Cdc42p-dependent MAP kinase pathway...

Hsp104 propagates the yeast prion [ PSI+ ], the infectious form of Sup35, by severing the prion seeds, but when Hsp104 is overexpressed, it cures [ PSI+ ] in a process that is not yet understood but may be caused by trimming, which removes monomers from the ends of the amyloid fibers. This curing was shown to depend on both the N-terminal domain of Hsp104 and the expression level of various members of the Hsp70 family, which raises the question as to whether these effects of Hsp70 are due to it binding to the Hsp70 binding site that was identified in the N-terminal domain of Hsp104, a site not involved in prion propagation...

The obligate intracellular pathogen Chlamydia trachomatis has unique metabolic requirements as it proceeds through its biphasic developmental cycle from within the inclusion within the host cell. In our previous study, we identified a host protein, PICALM, which localizes to the chlamydial inclusion. PICALM functions in many host pathways including the recycling of receptors, specific SNARE proteins, and molecules like transferrin, and maintaining cholesterol homeostasis. Hence, we hypothesized that PICALM functions to maintain the cholesterol content and to moderate trafficking from the endosomal recycling pathway to the inclusion, which controls chlamydial access to this pathway...

Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4 , which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F...