V-1 regulates capping protein activity in vivo.

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

9-cis Retinoic acid modulates myotrophin expression and its miR in physiological and pathophysiological cell models.

Functional studies indicate that essential cellular processes are controlled by Vitamin A derivatives. Among these the retinoic acid isoforms, all-trans- and 9-cis (9cRA), regulate the expression of various genes in both physiological and pathological conditions. Using several in vitro experimental models such as pancreatic ß-cells, pre-adipocytes and breast cancer cells with different phenotypes, we demonstrated the capability of 9cRA to modulate myotrophin (Mtpn) and miR-375 expressions. The 9cRA effect in pancreatic ß-cells line INS-1 832/13 point out a decreased expression of Mptn at both mRNA and protein levels associated to a concomitant increase of miR-375. We also studied the effect of this molecule on 3T3-L1 pre-adipocytes cells demonstrating a down-regulation of Mtpn and a dramatic increase of miR-375. Moreover, in the in vitro breast cancer model such as MDA-MB-231 and MCF-7 cells, 9cRA showed different effect on both Mtpn and miR-375 expression. In INS-1 832/13, 3T3-L1 pre-adipocytes and MCF-7 but not in MDA-MB-231, the effect of 9cRA on Mptn gene expression and its miR was under the control of RARs and RXRs receptors, as revealed by the exposure of these cell line to LE540 or HX603 receptor antagonists. In our findings 9cRA emerges has a hormone with a regulatory action on miR-375 that in most cases interfere with Mtpn expression.

Reconstitution of Arp2/3-Nucleated Actin Assembly with CP, V-1 and CARMIL.

Actin polymerization is often associated with membrane proteins containing capping-protein-interacting (CPI) motifs, such as CARMIL, CD2AP, and WASHCAP/Fam21. CPI motifs bind directly to actin capping protein (CP), and this interaction weakens the binding of CP to barbed ends of actin filaments, lessening the ability of CP to functionally cap those ends. The protein V-1 / myotrophin binds to the F-actin binding site on CP and sterically blocks CP from binding barbed ends. CPI-motif proteins also weaken the binding between V-1 and CP, which decreases the inhibitory effects of V-1, thereby freeing CP to cap barbed ends. Here, we address the question of whether CPI-motif proteins on a surface analogous to a membrane lead to net activation or inhibition of actin assembly nucleated by Arp2/3 complex. Using reconstitution with purified components, we discovered that CARMIL at the surface promotes and enhances actin assembly, countering the inhibitory effects of V-1 and thus activating CP. The reconstitution involves the presence of an Arp2/3 activator on the surface, along with Arp2/3 complex, V-1, CP, profilin and actin monomers in solution, recreating key features of cell physiology.

The ATO/miRNA-885-5p/MTPN axis induces reversal of drug-resistance in cholangiocarcinoma.

PURPOSE: Cholangiocarcinoma (CCA) is the second most malignant tumor of the hepatobiliary system. Due to its cumbersome early diagnosis and rapid progression, chemotherapy has become the main treatment option. Primary drug resistance is a major cause of the poor efficacy of chemotherapeutic drugs. Therefore, it is considered urgent to explore new drugs to overcome primary drug resistance of CCA. METHODS: Western blot and qRT-PCR assays were used to assess the expression of myotrophin (MTPN) and microRNA-885-5p (miR-885-5p) in CCA tissues and cells. The viability of CCA cells treated with arsenic trioxide (ATO), 5-fluorouracil (5-Fu) and cisplatin (CDDP) was analyzed using a CCK-8 assay. A luciferase reporter assay was used to assess the interaction between miR-885-5p and MTPN. Kaplan-Meier analyses were used for survival assessments. RESULT: We found that ATO can reduce the resistance of CCA cells to 5-Fu and CDDP and promote the killing effect of 5-Fu and CDDP. Low-dose ATO showed an anti-drug-resistance effect through up-regulation of the expression of miR-885-5p. Combined with sequencing results and database predictions, we found that MTPN may serve as a direct target of miR-885-5p. After MTPN knockdown, the sensitivity of CCA cells to 5-FU and CDDP was increased. Finally, we found that ATO can reverse chemotherapy resistance induced by overexpression of MTPN. CONCLUSION: Our data indicate that the ATO/miR-885-5p/MTPN axis may serve as a target for improving the sensitivity of CCA cells to chemotherapy.

Crystal structure of human V-1 in the apo form.

V-1, also known as myotrophin, is a 13 kDa ankyrin-repeat protein that binds and inhibits the heterodimeric actin capping protein (CP), which is a key regulator of cytoskeletal actin dynamics. The crystal structure of V-1 in complex with CP revealed that V-1 recognizes CP via residues spanning several ankyrin repeats. Here, the crystal structure of human V-1 is reported in the absence of the specific ligand at 2.3 Šresolution. In the asymmetric unit, the crystal contains two V-1 monomers that exhibit nearly identical structures (Cα r.m.s.d. of 0.47 Å). The overall structures of the two apo V-1 chains are also highly similar to that of CP-bound V-1 (Cα r.m.s.d.s of <0.50 Å), indicating that CP does not induce a large conformational change in V-1. Detailed structural comparisons using the computational program All Atom Motion Tree revealed that CP binding can be accomplished by minor side-chain rearrangements of several residues. These findings are consistent with the known biological role of V-1, in which it globally inhibits CP in the cytoplasm.

MiR-375, a microRNA related to diabetes.

MiR-375 is an important small non-coding RNA that is specifically expressed in islet cells of the pancreas. miR-375 is required for normal pancreatic genesis and influences not only ß-cell mass but also α-cell mass. miR-375 is also important to glucose-regulated insulin secretion through the regulation of the expression of Mtpn and Pdk1 genes. When human embryonic stem cells (hESCs) differentiate into endodermal lineages, miR-375 is highly expressed in the definitive endoderm, which suggests that miR-375 may have a distinct role in early development. miR-375 plays an important role in the complex regulatory network of pancreatic development, which could be regulated by pancreatic genes, such as NeuroD1, Ngn3, Pdx1 and Hnf6; additionally, miR-375 regulates genes related to pancreas development, cell growth and proliferation and insulin secretion genes to exert its function. Because of the special role of miR-375, it may be a potential target to treat diabetes. Antagonising miR-375 may enhance the effects of exendin-4 in patients, and controlling the expression of miR-375 could assist mature hESCs-derived ß-cells.

MRNA and miRNA expression patterns associated to pathways linked to metal mixture health effects.

Metals are a threat to human health by increasing disease risk. Experimental data have linked altered miRNA expression with exposure to some metals. MiRNAs comprise a large family of non-coding single-stranded molecules that primarily function to negatively regulate gene expression post-transcriptionally. Although several human populations are exposed to low concentrations of As, Cd and Pb as a mixture, most toxicology research focuses on the individual effects that these metals exert. Thus, this study aims to evaluate global miRNA and mRNA expression changes induced by a metal mixture containing NaAsO2, CdCl2, Pb(C2H3O2)2·3H2O and to predict possible metal-associated disease development under these conditions. Our results show that this metal mixture results in a miRNA expression profile that may be responsible for the mRNA expression changes observed under experimental conditions in which coding proteins are involved in cellular processes, including cell death, growth and proliferation related to the metal-associated inflammatory response and cancer.