Mechanical stretch increases L-type calcium channel stability in cardiomyocytes through a polycystin-1/AKT-dependent mechanism.

The L-type calcium channel (LTCC) is an important determinant of cardiac contractility. Therefore, changes in LTCC activity or protein levels could be expected to affect cardiac function. Several studies describing LTCC regulation are available, but only a few examine LTCC protein stability. Polycystin-1 (PC1) is a mechanosensor that regulates heart contractility and is involved in mechanical stretch-induced cardiac hypertrophy. PC1 was originally described as an unconventional Gi/o protein-coupled receptor in renal cells. We recently reported that PC1 regulates LTCC stability in cardiomyocytes under stress; however, the mechanism underlying this effect remains unknown. Here, we use cultured neonatal rat ventricular myocytes and hypo-osmotic stress (HS) to model mechanical stretch. The model shows that the Cavß2 subunit is necessary for LTCC stabilization in cardiomyocytes during mechanical stretch, acting through an AKT-dependent mechanism. Our data also shows that AKT activation depends on the G protein-coupled receptor activity of PC1, specifically its G protein-binding domain, and the associated Gßγ subunit of a heterotrimeric Gi/o protein. In fact, over-expression of the human PC1 C-terminal mutant lacking the G protein-binding domain blunted the AKT activation-induced increase in Cav1.2 protein in cardiomyocytes. These findings provide novel evidence that PC1 is involved in the regulation of cardiac LTCCs through a Gißγ-AKT-Cavß2 pathway, suggesting a new mechanism for regulation of cardiac function.

Cell death and survival through the endoplasmic reticulum-mitochondrial axis.

The endoplasmic reticulum has a central role in biosynthesis of a variety of proteins and lipids. Mitochondria generate ATP, synthesize and process numerous metabolites, and are key regulators of cell death. The architectures of endoplasmic reticulum and mitochondria change continually via the process of membrane fusion, fission, elongation, degradation, and renewal. These structural changes correlate with important changes in organellar function. Both organelles are capable of moving along the cytoskeleton, thus changing their cellular distribution. Numerous studies have demonstrated coordination and communication between mitochondria and endoplasmic reticulum. A focal point for these interactions is a zone of close contact between them known as the mitochondrial-associated endoplasmic reticulum membrane (MAM), which serves as a signaling juncture that facilitates calcium and lipid transfer between organelles. Here we review the emerging data on how communication between endoplasmic reticulum and mitochondria can modulate organelle function and determine cellular fate.

The 19S complex of the proteasome regulates nucleotide excision repair in yeast.

Previous studies suggest that the amino-terminal ubiquitin-like (ubl) domain of Rad23 protein can recruit the proteasome for a stimulatory role during nucleotide excision repair in the yeast Saccharomyces cerevisiae. In this report, we show that the 19S regulatory complex of the yeast proteasome can affect nucleotide excision repair independently of Rad23 protein. Strains with mutations in 19S regulatory subunits (but not 20S subunits) of the proteasome promote partial recovery of nucleotide excision repair in vivo in rad23 deletion mutants, but not in other nucleotide excision repair-defective strains tested. In addition, a strain that expresses a temperature-degradable ATPase subunit of the 19S regulatory complex manifests a dramatically increased rate of nucleotide excision repair in vivo. These data indicate that the 19S regulatory complex of the 26S proteasome can negatively regulate the rate of nucleotide excision repair in yeast and suggest that Rad23 protein not only recruits the 19S regulatory complex, but also can mediate functional interactions between the 19S regulatory complex and the nucleotide excision repair machinery. The 19S regulatory complex of the yeast proteasome functions in nucleotide excision repair independent of proteolysis.

Distinct roles of two binding sites for the bovine papillomavirus (BPV) E2 transactivator on BPV DNA replication.

The modulation of DNA replication by transcription factors was examined by using bovine papillomavirus type 1 (BPV). BPV replication in vivo requires two viral proteins: E1, an origin-binding protein, and E2, a transcriptional transactivator. In the origin, E1 interacts with a central region flanked by two binding sites for E2 (BS11 and BS12), of which only BS12 has been reported to be essential for replication in vivo. Using chemical interference and electrophoretic mobility shift assays, we found that the binding of E2 to each site stimulates the formation of distinct E1-origin complexes. A high-mobility C1 complex is formed by using critical E2 contacts to BS12 and E1 contacts to the dyad symmetry element. In contrast, interaction of E2 with the BS11 element on the other origin flank promotes the formation of the lower-mobility C3 complex. C3 is a novel species that resembles C2, a previously identified complex that is replication active and formed by E1 alone. The binding of E1 greatly differs in the C1 and C3 complexes, with E1 in the C1 complex limited to the origin dyad symmetry region and E1 in the C3 complex encompassing the region from the proximal edge of BS11 through the distal edge of BS12. We found that the presence of both E2-binding sites is necessary for wild-type replication activity in vivo, as well as for maximal production of the C3 complex. These results show that in the normal viral context, BS11 and BS12 play separate but synergetic roles in the initiation of viral DNA replication that are dependent on their location within the origin. Our data suggest a model in which the binding of E2 to each site sequentially stimulates the formation of distinct E1-origin complexes, leading to the replication-competent complex.

Induction of structural changes in the bovine papillomavirus type 1 origin of replication by the viral E1 and E2 proteins.

Chemical and enzymatic probing techniques were used to examine the interaction of the bovine papillomavirus type 1 E1 and E2 proteins with the viral origin of replication (ori). E1 was found to generate significant distortions to the structure of ori, as assayed by KMnO4 oxidation of DNA. The primary site of ori distortion was located within and adjacent to the AT-element of the core replicator sequence, although a number of minor structural transitions were also detected. The induction of these structural changes required ATP and appeared to require ATP hydrolysis. E2 was found to decrease the amount of E1 required for ori distortion but did not significantly alter the pattern of structural distortion. In contrast, the presence of E2 resulted in a biphasic mechanism for E1 binding to ori, as assayed by nuclease protection. Under these conditions, E1 bound preferentially to the dyad symmetry region containing the conserved Hpa I site. Higher levels of E1 were required for binding to the adjacent ori AT-rich region. Thus, these data suggest that E2 can order the stepwise binding of E1 to ori.

Organization of the human cholesteryl ester transfer protein gene.

The plasma cholesteryl ester transfer protein (CETP) catalyzes the transfer of phospholipids and neutral lipids between the lipoproteins. Thus, this protein may be important in modulating lipoprotein levels in the plasma. We have determined the primary structure and organization of the human CETP gene. Southern blotting of cellular DNA indicated a single copy of the CETP gene exists per haploid genome. Analysis of three overlapping genomic clones showed that the gene spans approximately 25 kbp and contains 16 exons (size range 32-250 bp). Overall, the sequence and organization of the CETP gene do not resemble those of other lipid-metabolizing enzymes or apolipoproteins. However, comparison of the CETP sequence, one exon at a time, with the sequences in the sequence databases revealed a striking identity of a pentapeptide sequence (ValLeuThrLeuAla) within the hydrophobic core of the signal sequences of human CETP, apolipoproteins A-IV and A-I, and lipoprotein lipase. This pentapeptide sequence was not found in the signal sequences of other proteins, suggesting that it may mediate a specialized function related to lipid metabolism or transport.