CFTR Haplotype Analysis Reveals Genetic Heterogeneity in the Etiology of Congenital Bilateral Aplasia of the Vas Deferens

Congenital bilateral aplasia of the vas deferens (CBAVD) was suggested to be a mild form of cystic fibrosis (CF). Mutation analysis of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in males with CBAVD revealed that in some males CBAVD is caused by two defective CFTR alleles. The genetic basis of CBAVD in the other males and its association with CF remained unclear. We undertook this study to test the hypothesis of commonality of CBAVD and CF by haplotype analysis, in the CFTR locus, of males suffering from CBAVD and of their families. According to the hypothesis of commonality of CBAVD and CF, two brothers with CBAVD are expected to carry the same two CFTR alleles, while their fertile brothers are expected to carry at least one different allele. Eleven families were studied, of which two families, with unidentified CFTR mutations, did not support this hypothesis. In these families two brothers with CBAVD inherited different CFTR alleles. Their fertile brothers inherited the same CFTR alleles as their brothers with CBAVD. These results provide evidence for genetic heterogeneity in CBAVD. Though in some families CBAVD is associated with two CFTR mutations, we suggest that in others it is caused by other mechanisms, such as mutations at other loci or homozygosity or heterozygosity for partially penetrant CFTR mutations.     

Highly variable incidence of cystic fibrosis and different mutation distribution among different Jewish ethnic groups in Israel

The incidence of cystic fibrosis (CF) and the frequency of disease-causing mutations varies among different ethnic and geographic populations. The Jewish population around the world is comprised of two major ethnic groups; Ashkenazi and non-Ashkenazi. The latter is further classified according to country of origin. In this study, we analyzed the incidence of CF and the distribution of CF mutations in the general Jewish population in Israel and in most of the Jewish ethnic subgroups. The disease frequency varies considerably among the latter. Among Ashkenazi Jews, the frequency of CF is 13300, which is similar to the frequency in most Caucasian populations. Among non-Ashkenazi Jews, the disease occurs at a similar frequency among Jews from Libya (12700), Georgia (12700), Greece and Bulgaria (12400), but is rare in Jews from Yemen (18800), Morocco (115000), Iraq (132000), and Iran (139000). So far, only 12 mutations have been identified in Israeli Jews, and this enables the identification of 91% of the CF chromosomes in the entire Jewish CF population. However, in each Jewish ethnic group, the disease is caused by a different repertoire of mutations. The frequency of identified mutations is high in Ashkenazi Jews (95%), and in Jews originating from Tunisia (100%), Libya (91%), Turkey (90%), and Georgia (88%). However, a lower frequency of mutations can be identified in Moroccan (85%), Egyptian (50%), and Yemenite (0%) Jews. For genetic counseling of a Jewish individual, it is necessary to calculate the residual risk according to ethnic origin. Carrier screening of healthy Jewish individuals is currently feasible for Ashkenazi Tunisian, Libyan, Turkish, and Georgian Jews. These results provide the required information for genetic counseling of Jewish CF families and screening programs of Jewish populations worldwide.     

In a randomized trial in prostate cancer patients,dietary protein restriction modifies markers of leptin and insulin signaling in plasma extracellular vesicles

Obesity, metabolic syndrome, and hyperleptinemia are associated with aging and age‐associated diseases including prostate cancer. One experimental approach to inhibit tumor growth is to reduce dietary protein intake and hence levels of circulating amino acids. Dietary protein restriction (PR) increases insulin sensitivity and suppresses prostate cancer cell tumor growth in animal models, providing a rationale for clinical trials. We sought to demonstrate that biomarkers derived from plasma extracellular vesicles (EVs) reflect systemic leptin and insulin signaling and respond to dietary interventions. We studied plasma samples from men with prostate cancer awaiting prostatectomy who participated in a randomized trial of one month of PR or control diet. We found increased levels of leptin receptor in the PR group in total plasma EVs and in a subpopulation of plasma EVs expressing the neuronal marker L1CAM. Protein restriction also shifted the phosphorylation status of the insulin receptor signal transducer protein IRS1 in L1CAM+ EVs in a manner suggestive of improved insulin sensitivity. Dietary PR modifies indicators of leptin and insulin signaling in circulating EVs. These findings are consistent with improved insulin and leptin sensitivity in response to PR and open a new window for following physiologic responses to dietary interventions in humans.     

tBID Homooligomerizes in the mitochondrial membrane to induce apoptosis.

Activation of the tumor necrosis factor R1/Fas receptor results in the cleavage of cytosolic BID to truncated tBID. tBID translocates to the mitochondria to induce the oligomerization of BAX or BAK, resulting in the release of cytochrome c (Cyt c). Here we demonstrate that in tumor necrosis factor alpha-activated FL5.12 cells, tBID becomes part of a 45-kDa cross-linkable mitochondrial complex that does not include BAX or BAK. Using fluorescence resonance energy transfer analysis and co-immunoprecipitation, we demonstrate that tBID-tBID interactions occur in the mitochondria of living cells. Cross-linking experiments using a tBID-GST chimera indicated that tBID forms homotrimers in the mitochondrial membrane. To test the functional consequence of tBID oligomerization, we expressed a chimeric FKBP-tBID molecule. Enforced dimerization of FKBP-tBID by the bivalent ligand FK1012 resulted in Cyt c release, caspase activation, and apoptosis. Surprisingly, enforced dimerization of tBID did not result in the dimerization of either BAX or BAK. Moreover, a tBID BH3 mutant (G94E), which does not interact with or induce the dimerization of either BAX or BAK, formed the 45-kDa complex and induced both Cyt c release and apoptosis. Thus, tBID oligomerization may represent an alternative mechanism for inducing mitochondrial dysfunction and apoptosis.     

Morphine induces desensitization of insulin receptor signaling

Morphine analgesia is mediated principally by the micro -opioid receptor (MOR). Since morphine and other opiates have been shown to influence glucose homeostasis, we investigated the hypothesis of direct cross talk between the MOR and the insulin receptor (IR) signaling cascades. We show that prolonged morphine exposure of cell lines expressing endogenous or transfected MOR, IR, and the insulin substrate 1 (IRS-1) protein specifically desensitizes IR signaling to Akt and ERK cascades. Morphine caused serine phosphorylation of the IR and impaired the formation of the signaling complex among the IR, Shc, and Grb2. Morphine also resulted in IRS-1 phosphorylation at serine 612 and reduced tyrosine phosphorylation at the YMXM p85-binding motifs, weakening the association of the IRS-1/p85 phosphatidylinositol 3-kinase complex. However, the IRS-1/Grb2 complex was unaffected by chronic morphine treatment. These results suggest that morphine attenuates IR signaling to Akt by disrupting the IRS-1-p85 interaction but inhibits signaling to ERK by disruption of the complex among the IR, Shc, and Grb2. Finally, we show that systemic morphine induced IRS-1 phosphorylation at Ser612 in the hypothalamus and hippocampus of wild type, but not MOR knockout, mice. Our results demonstrate that opiates can inhibit insulin signaling through direct cross talk between the downstream signaling pathways of the MOR and the IR.     

β-Synuclein occurs in vivo in lipid-associated oligomers and forms hetero-oligomers with α-synuclein

α-synuclein (αS) and β-synuclein (βS) are homologous proteins implicated in Parkinson's disease and related synucleinopathies. While αS is neurotoxic and its aggregation and deposition in Lewy bodies is related to neurodegeneration, βS is considered as a potent inhibitor of αS aggregation and toxicity. No mechanism for the neuroprotective role of βS has been described before. Here, we report that similar to αS, βS normally occurs in lipid-associated, soluble oligomers in wild-type (WT) mouse brains. We partially purified βS and αS proteins from whole mouse brain by size exclusion followed by ion exchange chromatography and found highly similar elution profiles. Using this technique, we were able to partially separate βS from αS and further separate βS monomer from its own oligomers. Importantly, we show that although αS and βS share high degree of similarities, βS oligomerization is not affected by increasing cellular levels of polyunsaturated fatty acids (PUFAs), while αS oligomerization is dramatically enhanced by PUFA. We show the in vivo occurrence of hetero-oligomers of αS and βS and suggest that βS expression inhibits PUFA-enhanced αS oligomerization by forming hetero-oligomers up to a quatramer that do not further propagate.     

A Promiscuous Conformational Switch in the Secondary Multidrug Transporter MdfA

Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter. Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active transport is triggered promiscuously by merely occupying the Mdr-binding site.     

Gas2l3, a Novel Constriction Site-Associated Protein Whose Regulation Is Mediated by the APC/CCdh1 Complex

Growth arrest-specific 2-like protein 3 (Gas2l3) was recently identified as an Actin/Tubulin cross-linker protein that regulates cytokinesis. Using cell-free systems from both frog eggs and human cells, we show that the Gas2l3 protein is targeted for ubiquitin-mediated proteolysis by the APC/C^Cdh1 complex, but not by the APC/C^Cdc20 complex, and is phosphorylated by Cdk1 in mitosis. Moreover, late in cytokinesis, Gas2l3 is exclusively localized to the constriction sites, which are the narrowest parts of the intercellular bridge connecting the two daughter cells. Overexpression of Gas2l3 specifically interferes with cell abscission, which is the final stage of cell division, when the cutting of the intercellular bridge at the constriction sites occurs. We therefore suggest that Gas2l3 is part of the cellular mechanism that terminates cell division.