Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Ubiquitin-proteasome dependent regulation of Profilin2 (Pfn2) by a cellular inhibitor of apoptotic protein 1 (cIAP1).

The two major isoforms of the profilin (Pfn) family of proteins in mammals are Pfn1 and Pfn2. Pfn1 is a universal actin cytoskeletal regulator, while Pfn2 is an actin binding protein and mediator of synapse architecture, specific to neural tissues. However, it has recently been suggested that Pfn2 is also widely distributed in various tissues and involved in numerous cellular events as well as cytoskeletal regulation. In our previous study, we showed that Pfn1 is regulated by carboxyl terminus of Hsc70-Interacting Protein (CHIP) via an ubiquitin (Ub) proteasome system; although, the mechanism of regulation of Pfn2 is unknown. In this report, we demonstrate that Pfn2 is heavily ubiquitinated via differential Ub-linkages for degradation or as a regulatory signal. We also show that cellular inhibitor of apoptosis 1 (cIAP1) rather than CHIP, functions as an E3 ligase that targets Pfn2 for proteasomal degradation. Finally, we observed that Pfn2 levels, regulated by cIAP1, affected intracellular levels of reactive oxygen species. These results may provide a regulatory mechanism for cellular function of Pfn2 in various tissues.

Regulation of fragile X mental retardation 1 protein by C-terminus of Hsc70-interacting protein depends on its phosphorylation status.

The fragile X mental retardation 1 (FMR1) protein binds mRNA and acts as a negative regulator of translation. Lack of FMR1 causes the most common neurological disorder, fragile X syndrome, while its overexpression is associated with metastasis of breast cancer. Its activity has been well-studied in nervous tissue, but recent evidence as well as its role in cancer indicates that it also acts in other tissues. We have investigated the expression of FMR1 in brain and other tissues of mouse and examined its regulation. We detected expression of FMR1 in liver and heart tissues of mice as well as in brain tissue, supporting other contentions that it acts in non-nervous tissue. Expression of FMR1 inversely correlated with expression of the C-terminus of Hsc70-interacting protein (CHIP) and, based on the known activity of CHIP in protein homeostasis, we suggest that CHIP regulates expression of FMR1. CHIP ubiquitinated FMR1 for proteasomal degradation in a molecular chaperone-independent manner. FMR1 expression was reduced following treatment with okadaic acid, a phosphatase inhibitor, but not in CHIP-depleted cells. Also, a non-phospho FMR1 mutant was much less efficiently ubiquitinated by CHIP and had a longer half-life compared to either wild-type FMR or a phospho-mimic mutant. Taken together, our results demonstrate that CHIP regulates the levels of FMR1 as an E3 ubiquitin ligase in phosphorylation-dependent manner, suggesting that CHIP regulates FMR1-mediated translational repression by regulating the levels of FMR1.

C-Terminus of Hsc70-interacting protein regulates profilin1 and breast cancer cell migration.

Profilin1 (Pfn1) is a key mediator of actin polymerization and regulates cell migration. Low expression of Pfn1 is implicated in tumorigenesis of various cancers, including breast cancer. The regulatory mechanism behind Pfn1 levels has not yet been elucidated. In the present study, we find that Pfn1 is poly-ubiquitinated in human cell lines, and a portion of poly-ubiquitinated Pfn1 is regulated in a proteasome-dependent manner. C-terminus of Hsc70-interacting protein (CHIP), a co-chaperone E3 ligase, interacts with and ubiquitinates Pfn1, targeting it for proteasome-dependent degradation. Depletion of CHIP stabilizes Pfn1, suggesting that CHIP functions as a major E3 ligase for Pfn1. Stable expression of wild-type CHIP in the breast cancer cell line MDA-MB231 yielded downregulation of Pfn1 and enhanced cell migration. Pfn1 overexpression in MDA-MB231 cells expressing wild-type CHIP suppressed the enhanced cell migration. Taken together, our results demonstrate that CHIP regulates Pfn1 levels as an E3 ligase, and possibly plays a role in cell migration and metastasis of breast cancer.

Nuclear endonuclease G controls cell proliferation in ovarian cancer.

Ovarian cancer is characterized by a high degree of genetic heterogeneity. Platinum-based chemotherapy and some gene-targeted therapies have shown limited treatment efficacy due to toxicity and recurrence, and thus, it is essential to identify additional therapeutic targets based on an understanding of the pathological mechanism. Here, we report that endonuclease G, which exhibits altered expression in ovarian cancer, does not function as a cell death effector that digests chromosomal DNA in ovarian cancer. Endonuclease G is modulated by intracellular reactive oxygen species dynamics and plays a role in cell proliferation in ovarian cancer, suggesting that targeting endonuclease G alone or in combination with other antitumor agents may have the potential for development into a treatment for endonuclease G-overexpressing cancers, including ovarian cancer.

A novel function of cIAP1 as a mediator of CHIP-driven eIF4E regulation.

eIF4E is an initiator protein in cap-dependent translation. Its overexpression is linked to tumorigenesis in various human cancers, suggesting that the levels of eIF4E must be under tight control in normal cells. Although several eIF4E regulatory mechanisms have been demonstrated, the intracellular mechanisms controlling eIF4E protein levels remain poorly understood. Here, we report that eIF4E is efficiently regulated by dual mechanisms, both involving human inhibitor of apoptosis family protein cIAP1. cIAP1 itself ubiquitinates eIF4E as an E3 ligase, and interestingly, cIAP1 also functions as a mediator to present eIF4E to another E3 ligase, CHIP. This collaborative activity of cIAP1 and CHIP directs eIF4E toward degradation, controlling its levels and suppressing tumorigenesis. Our results provide the first evidence for a mediator function of cIAP1 and collaborative activity of cIAP1 and CHIP, suggesting that maintaining balanced levels of these E3 ligases might be beneficial for normal cell growth.

Increased alternative lengthening of telomere phenotypes of telomerase-negative immortal cells upon trichostatin--a treatment.

Human immortal cells maintain their telomeres either by telomerase or by alternative lengthening of telomeres (ALT) that is based on homologous telomeric recombination. Previous studies showed that the ALT mechanism is activated in non-ALT cells when heterochromatic features are reduced. In this study, we examined the ALT phenotypes of ALT cells after treatment with trichostatin-A (TSA), which is an inhibitor of histone deacetylases and causes global chromatin decondensation. The ALT cells remained telomerase-negative after TSA treatment. ALT-associated promyelocytic leukemia (PML) nuclear bodies and telomere sister chromatid exchanges, typical ALT phenotypes, markedly increased in the TSA-treated cells, while the telomere length remained unchanged. In addition, telomerase expression in the ALT cells suppressed TSA-mediated ALT phenotype enhancement. Our results show that certain ALT phenotypes become more pronounced when chromatin is decondensed, and also suggest that the ALT mechanism may compete with telomerase for telomere maintenance in cells that lack heterochromatin.

Identification of cdc25 gene in pinewood nematode, Bursaphelenchus xylophilus, and its function in reproduction.

The cdc25 gene, which is highly conserved in many eukaryotes, encodes a phosphatase that plays essential roles in cell cycle regulation. We identified a cdc25 ortholog in the pinewood nematode, Bursaphelenchus xylophilus. The B. xylophilus ortholog (Bx-cdc25) was found to be highly similar to Caenorhabditis elegans cdc-25.2 in sequence as well as in gene structure, both having long intron 1. The Bx-cdc25 gene was determined to be composed of seven exons and six introns in a 2,580 bp region, and was shown to encode 360 amino acids of a protein containing a highly-conserved phosphatase domain. Bx-cdc25 mRNA was hardly detectable throughout the juvenile stages but was highly expressed in eggs and in both female and male adults. Functional conservation during germline development between C. elegans cdc25 and Bx-cdc25 was revealed by Bx-cdc25 RNA interference in C. elegans.