Cross-talk between mutant p53 and p62/SQSTM1 augments cancer cell migration by promoting the degradation of cell adhesion proteins.

Missense mutations in the p53 tumor suppressor abound in human cancer. Common ("hotspot") mutations endow mutant p53 (mutp53) proteins with oncogenic gain of function (GOF), including enhanced cell migration and invasiveness, favoring cancer progression. GOF is usually attributed to transcriptional effects of mutp53. To elucidate transcription-independent effects of mutp53, we characterized the protein interactome of the p53R273H mutant in cells derived from pancreatic ductal adenocarcinoma (PDAC), where p53R273H is the most frequent p53 mutant. We now report that p53R273H, but not the p53R175H hotspot mutant, interacts with SQSTM1/p62 and promotes cancer cell migration and invasion in a p62-dependent manner. Mechanistically, the p53R273H-p62 axis drives the proteasomal degradation of several cell junction­associated proteins, including the gap junction protein Connexin 43, facilitating scattered cell migration. Concordantly, down-regulation of Connexin 43 augments PDAC cell migration, while its forced overexpression blunts the promigratory effect of the p53R273H-p62 axis. These findings define a mechanism of mutp53 GOF.

Identification and characterization of signal peptide of Mitofusin1 (Mfn1).

Mitofusin1 (Mfn1) mediates outer mitochondrial membrane (OMM) fusion in Opisthokonts. The uncharacterized TM comprises to two helices (namely, the TM1 and TM2) connected by an intermembrane loop. Consistent with previous studies, our results from in silico analyses show that all mitofusins lack N terminal-MTS and the TM may act an internal MTS. We have identified a conserved region in TM domain that is responsible for mitochondrial localization of Mfn1/2. Thus, our results suggest the dual function of TM; in OMM anchoring and signaling Mfn1 to mitochondria. Our study illuminates the underlying role of TM for mitochondrial localization of Mfn1 on one hand and also paves a way for the development of tools for in silico prediction of cellular localization of proteins.

Ca2+ as a coordinator of skeletal muscle differentiation, fusion and contraction.

Muscle regeneration is essential for vertebrate muscle homeostasis and recovery after injury. During regeneration, muscle stem cells differentiate into myocytes, which then fuse with pre-existing muscle fibres. Hence, differentiation, fusion and contraction must be tightly regulated during regeneration to avoid the disastrous consequences of premature fusion of myocytes to actively contracting fibres. Cytosolic calcium (Ca2+ ), which is coupled to both induction of myogenic differentiation and contraction, has more recently been implicated in the regulation of myocyte-to-myotube fusion. In this viewpoint, we propose that Ca2+ -mediated coordination of differentiation, fusion and contraction is a feature selected in the amniotes to facilitate muscle regeneration.

ERK1/2 inhibition promotes robust myotube growth via CaMKII activation resulting in myoblast-to-myotube fusion.

Myoblast fusion is essential for muscle development and regeneration. Yet, it remains poorly understood how mononucleated myoblasts fuse with preexisting fibers. We demonstrate that ERK1/2 inhibition (ERKi) induces robust differentiation and fusion of primary mouse myoblasts through a linear pathway involving RXR, ryanodine receptors, and calcium-dependent activation of CaMKII in nascent myotubes. CaMKII activation results in myotube growth via fusion with mononucleated myoblasts at a fusogenic synapse. Mechanistically, CaMKII interacts with and regulates MYMK and Rac1, and CaMKIIδ/γ knockout mice exhibit smaller regenerated myofibers following injury. In addition, the expression of a dominant negative CaMKII inhibits the formation of large multinucleated myotubes. Finally, we demonstrate the evolutionary conservation of the pathway in chicken myoblasts. We conclude that ERK1/2 represses a signaling cascade leading to CaMKII-mediated fusion of myoblasts to myotubes, providing an attractive target for the cultivated meat industry and regenerative medicine.

Molecular Evolution and Diversification of Proteins Involved in miRNA Maturation Pathway.

Small RNAs (smRNA, 19-25 nucleotides long), which are transcribed by RNA polymerase II, regulate the expression of genes involved in a multitude of processes in eukaryotes. miRNA biogenesis and the proteins involved in the biogenesis pathway differ across plant and animal lineages. The major proteins constituting the biogenesis pathway, namely, the Dicers (DCL/DCR) and Argonautes (AGOs), have been extensively studied. However, the accessory proteins (DAWDLE (DDL), SERRATE (SE), and TOUGH (TGH)) of the pathway that differs across the two lineages remain largely uncharacterized. We present the first detailed report on the molecular evolution and divergence of these proteins across eukaryotes. Although DDL is present in eukaryotes and prokaryotes, SE and TGH appear to be specific to eukaryotes. The addition/deletion of specific domains and/or domain-specific sequence divergence in the three proteins points to the observed functional divergence of these proteins across the two lineages, which correlates with the differences in miRNA length across the two lineages. Our data enhance the current understanding of the structure-function relationship of these proteins and reveals previous unexplored crucial residues in the three proteins that can be used as a basis for further functional characterization. The data presented here on the number of miRNAs in crown eukaryotic lineages are consistent with the notion of the expansion of the number of miRNA-coding genes in animal and plant lineages correlating with organismal complexity. Whether this difference in functionally correlates with the diversification (or presence/absence) of the three proteins studied here or the miRNA signaling in the plant and animal lineages is unclear. Based on our results of the three proteins studied here and previously available data concerning the evolution of miRNA genes in the plant and animal lineages, we believe that miRNAs probably evolved once in the ancestor to crown eukaryotes and have diversified independently in the eukaryotes.

Molecular evolution of proteins mediating mitochondrial fission-fusion dynamics.

Eukaryotes employ a subset of dynamins to mediate mitochondrial fusion and fission dynamics. Here we report the molecular evolution and diversification of the dynamin-related mitochondrial proteins that drive the fission (Drp1) and the fusion processes (mitofusin and OPA1). We demonstrate that the three paralogs emerged concurrently in an early mitochondriate eukaryotic ancestor. Furthermore, multiple independent duplication events from an ancestral bifunctional fission protein gave rise to specialized fission proteins. The evolutionary history of these proteins is marked by transformations that include independent gain and loss events occurring at the levels of entire genes, specific functional domains, and intronic regions. The domain level variations primarily comprise loss-gain of lineage specific domains that are present in the terminal regions of the sequences.

Molecular evolution guided functional analyses reveals Nucleobindin-1 as a canonical E-box binding protein promoting Epithelial-to-Mesenchymal transition (EMT).

Calcium binding proteins (CBPs) function in response to changes in intracellular calcium (Ca2+) levels by modulating intracellular signaling pathways. Calcium sensors, including Nucleobindins (Nucb1/2) undergo Ca2+-binding induced conformational changes and bind to target proteins. Nucleobindins possess additional uncharacterized domains including partly characterized EF-hands. We study the molecular evolution of Nucleobindins in eukaryotes emphasizing on the N-terminal DNA binding domain (DBD) that emerged as a result of domain insertion event in Nucb1/2 domain-scaffold in an ancestor to the opisthokonts. Our results from in silico analyses and functional assays revealed that DBD of Nucb1 binds to canonical E-box sequences and triggers cell epithelial-mesenchymal transition (EMT). Thus, post gene duplication, Nucb1 has emerged as unconventional Ca2+-binding transcriptional regulators that can induce EMT.