No abstract

The surface expression and channel activation of transient receptor potential canonical 6 (TRPC6) were regulated by tyrosine phosphorylation and resultant binding with stimulatory PLC-γ1 and inhibitory nephrin. Disease-causing mutations made the TRPC6s insensitive to nephrin suppression, suggesting that the cell-type–specific regulation of TRPC6 might be involved in the pathogenesis.

Leber congenital amaurosis (LCA) is a hereditary early-onset retinal dystrophy that is accompanied by severe macular degeneration. In this study, novel compound heterozygous mutations were identified as LCA-causative in chaperonin-containing TCP-1, subunit 2 (CCT2), a gene that encodes the molecular chaperone protein, CCTβ. The zebrafish mutants of CCTβ are known to exhibit the eye phenotype while its mutation and association with human disease have been unknown. The CCT proteins (CCT α-θ) forms ring complex for its chaperon function. The LCA mutants of CCTβ, T400P and R516H, are biochemically instable and the affinity for the adjacent subunit, CCTγ, was affected distinctly in both mutants. The patient-derived induced pluripotent stem cells (iPSCs), carrying these CCTβ mutants, were less proliferative than the control iPSCs. Decreased proliferation under Cct2 knockdown in 661W cells was significantly rescued by wild-type CCTβ expression. However, the expression of T400P and R516H didn’t exhibit the significant effect. In mouse retina, both CCTβ and CCTγ are expressed in the retinal ganglion cells and connecting cilium of photoreceptor cells. The Cct2 knockdown decreased its major client protein, transducing β1 (Gβ1). Here we report the novel LCA mutations in CCTβ and the impact of chaperon disability by these mutations in cellular biology.

A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors). Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin. In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain. This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M r of 46,000 (p46). From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK). Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46. Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation. On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated. The enzymatic activity per se of PGK did not seem to be required for its activity. West-Western blot analysis showed that PGK could directly interact with tubulin. These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat (DPR) proteins. Although it has been reported that DPR proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we have first confirmed that proline–arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity and found that the poly-PR-induced neurotoxicity is repressed by the acceleration of ribosomal RNA synthesis. These results suggest that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. We have further identified DEAD-box RNA helicases as poly-PR-binding proteins, the functions of which are inhibited by poly-PR. The enforced reduction in the expression of DEAD-box RNA helicases causes impairment of ribosome biogenesis and neuronal cell death. These results together suggest that poly-PR causes neurotoxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

The serine/threonine kinase mTOR forms two distinct complexes, mTORC1 and mTORC2, and controls a number of biological processes, including proliferation, survival and autophagy. Although the function of mTORC1 has been extensively studied, the mTORC2 signaling pathway largely remains to be elucidated. Here, we have shown that mTORC2 phosphorylates filamin A, an actin cross-linking protein, at serine 2152 (S2152) both in vivo and in living cells. Treatment of HeLa cells with Torin1 (an mTORC1/mTORC2 inhibitor), but not rapamycin (an mTORC1 inhibitor), suppressed the phosphorylation of filamin A, which decreased the binding of filamin A with b7-integrin cytoplasmic tail. Torin1 also inhibited focal adhesion formation and cell migration in A7 filamin A-replete melanoma cells but not in M2 filamin A-deficient cells, suggesting a pivotal role for mTORC2 in filamin A function. Finally, reduced focal adhesion formation in M2 cells was significantly rescued by expressing wild type but not S2152A nonphosphorylatable mutant of filamin A. Taken together, our results indicate that mTORC2 regulates filamin A-dependent focal adhesions and cell migration.

The wild-type and mutant derivatives of the integrase protein of feline immunodeficiency virus (FIV) were cloned and expressed in Escherichia coli. The purified proteins were examined using various model DNA substrates for their catalytic activities: 3'-end processing, 3'-end joining, and disintegration. The reactions required the presence of either Mn2+ or Mg2+ as a divalent cation. The N-terminal and C-subterminal domains (residues 1-52 and 189-235, respectively) were necessary for 3'-end processing and joining reactions but not for disintegration. Substitution of asparagine for the highly conserved aspartic acid at position 118 resulted in a complete loss of all three activities, confirming that the catalytic domain resides in the central core region (residues 53-188) of the protein. Deletion of the C-terminus (residues 236-281) resulted in a FIV integrase mutant that had efficient 3'-end processing and disintegration activities but weak 3'-end joining activity, a finding that has not been reported previously with other retroviral integrases. The result suggests that the C-terminus is the primary binding site for target DNA. Attachment of a histidine-tag at the N-terminus of the wild-type and deletion derivatives increased the binding affinity to the DNA substrate, resulting in altered levels of catalytic activities and selection of integration sites. Similar to other retroviral integrases, certain pairs of mutant derivatives of FIV integrase could complement each other to restitute 3'-end processing and joining activities, suggesting that formation of functional multimers is a general feature of proteins in the integrase family.