Vps13D Encodes a Ubiquitin-Binding Protein that Is Required for the Regulation of Mitochondrial Size and Clearance.

The clearance of mitochondria by autophagy, mitophagy, is important for cell and organism health [1], and known to be regulated by ubiquitin. During Drosophila intestine development, cells undergo a dramatic reduction in cell size and clearance of mitochondria that depends on autophagy, the E1 ubiquitin-activating enzyme Uba1, and ubiquitin [2]. Here we screen a collection of putative ubiquitin-binding domain-encoding genes for cell size reduction and autophagy phenotypes. We identify the endosomal sorting complex required for transport (ESCRT) components TSG101 and Vps36, as well as the novel gene Vps13D. Vps13D is an essential gene that is necessary for autophagy, mitochondrial size, and mitochondrial clearance in Drosophila. Interestingly, a similar mitochondrial phenotype is observed in VPS13D mutant human cells. The ubiquitin-associated (UBA) domain of Vps13D binds K63 ubiquitin chains, and mutants lacking the UBA domain have defects in mitochondrial size and clearance and exhibit semi-lethality, highlighting the importance of Vps13D ubiquitin binding in both mitochondrial health and development. VPS13D mutant cells possess phosphorylated DRP1 and mitochondrial fission factor (MFF) as well as DRP1 association with mitochondria, suggesting that VPS13D functions downstream of these known regulators of mitochondrial fission. In addition, the large Vps13D mitochondrial and cell size phenotypes are suppressed by decreased mitochondrial fusion gene function. Thus, these results provide a previously unknown link between ubiquitin, mitochondrial size regulation, and autophagy.

Uba1 functions in Atg7- and Atg3-independent autophagy.

Autophagy is a conserved process that delivers components of the cytoplasm to lysosomes for degradation. The E1 and E2 enzymes encoded by Atg7 and Atg3 are thought to be essential for autophagy involving the ubiquitin-like protein Atg8. Here, we describe an Atg7- and Atg3-independent autophagy pathway that facilitates programmed reduction of cell size during intestine cell death. Although multiple components of the core autophagy pathways, including Atg8, are required for autophagy and cells to shrink in the midgut of the intestine, loss of either Atg7 or Atg3 function does not influence these cellular processes. Rather, Uba1, the E1 enzyme used in ubiquitylation, is required for autophagy and reduction of cell size. Our data reveal that distinct autophagy programs are used by different cells within an animal, and disclose an unappreciated role for ubiquitin activation in autophagy.

Atg6 is required for multiple vesicle trafficking pathways and hematopoiesis in Drosophila.

Atg6 (beclin 1 in mammals) is a core component of the Vps34 complex that is required for autophagy. Beclin 1 (Becn1) functions as a tumor suppressor, and Becn1(+/-) tumors in mice possess elevated cell stress and p62 levels, altered NF-κB signaling and genome instability. The tumor suppressor function of Becn1 has been attributed to its role in autophagy, and the potential functions of Atg6/Becn1 in other vesicle trafficking pathways for tumor development have not been considered. Here, we generate Atg6 mutant Drosophila and demonstrate that Atg6 is essential for autophagy, endocytosis and protein secretion. By contrast, the core autophagy gene Atg1 is required for autophagy and protein secretion, but it is not required for endocytosis. Unlike null mutants of other core autophagy genes, all Atg6 mutant animals possess blood cell masses. Atg6 mutants have enlarged lymph glands (the hematopoietic organ in Drosophila), possess elevated blood cell numbers, and the formation of melanotic blood cell masses in these mutants is not suppressed by mutations in either p62 or NFκB genes. Thus, like mammals, altered Atg6 function in flies causes hematopoietic abnormalities and lethality, and our data indicate that this is due to defects in multiple membrane trafficking processes.

The centrosome regulates the Rab11- dependent recycling endosome pathway at appendages of the mother centriole.

The recycling endosome localizes to a pericentrosomal region via microtubule-dependent transport. We previously showed that Sec15, an effector of the recycling endosome component, Rab11-GTPase, interacts with the mother centriole appendage protein, centriolin, suggesting an interaction between endosomes and centrosomes. Here we show that the recycling endosome associates with the appendages of the mother (older) centriole. We show that two mother centriole appendage proteins, centriolin and cenexin/ODF2, regulate association of the endosome components Rab11, the Rab11 GTP-activating protein Evi5, and the exocyst at the mother centriole. Development of an in vitro method for reconstituting endosome protein complexes onto isolated membrane-free centrosomes demonstrates that purified GTP-Rab11 but not GDP-Rab11 binds to mother centriole appendages in the absence of membranes. Moreover, centriolin depletion displaces the centrosomal Rab11 GAP, Evi5, and increases mother-centriole-associated Rab11; depletion of Evi5 also increases centrosomal Rab11. This indicates that centriolin localizes Evi5 to centriolar appendages to turn off centrosomal Rab11 activity. Finally, centriolin depletion disrupts recycling endosome organization and function, suggesting a role for mother centriole proteins in the regulation of Rab11 localization and activity at the mother centriole.

Genetic susceptibility to coronary heart disease in type 2 diabetes: 3 independent studies.

OBJECTIVES: The aim of this study was to evaluate whether coronary heart disease (CHD)-susceptibility loci identified by genome-wide association studies of the general population also contribute to CHD in type 2 diabetes. BACKGROUND: No study has examined the effects of these genetic variants on CHD in diabetic patients. METHODS: We genotyped 15 genetic markers of 12 loci in 3 studies of diabetic patients: the prospective Nurses' Health Study (309 CHD cases, and 544 control subjects) and Health Professional Follow-up Study (345 CHD cases, and 451 control subjects) and the cross-sectional Joslin Heart Study (422 CHD cases, and 435 control subjects). RESULTS: Five single-nucleotide polymorphisms, rs4977574 (CDKN2A/2B), rs12526453 (PHACTR1), rs646776 (CELSR2-PSRC1-SORT1), rs2259816 (HNF1A), and rs11206510 (PCSK9) showed directionally consistent associations with CHD in the 3 studies, with combined odds ratios (ORs) ranging from 1.17 to 1.25 (p = 0.03 to 0.0002). None of the other single-nucleotide polymorphisms reached significance in individual or combined analyses. A genetic risk score (GRS) was created by combining the risk alleles of the 5 significantly associated loci. The OR of CHD/GRS unit was 1.19 (95% confidence interval: 1.13 to 1.26; p < 0.0001). Individuals with GRS ≥8 (19% of diabetic subjects) had almost a 2-fold increase in CHD risk (OR: 1.94, 95% confidence interval: 1.60 to 2.35) as compared with individuals with GRS ≤5 (30% of diabetic subjects). Prediction of CHD was significantly improved (p < 0.001) when the GRS was added to a model including clinical predictors in the combined samples. CONCLUSIONS: Our results illustrate the consistency and differences in the determinants of genetic susceptibility to CHD in diabetic patients and the general populations.

Exploiting the mitochondrial unfolded protein response for cancer therapy in mice and human cells.

Fine tuning of the protein folding environment in subcellular organelles, such as mitochondria, is important for adaptive homeostasis and may participate in human diseases, but the regulators of this process are still largely elusive. Here, we have shown that selective targeting of heat shock protein-90 (Hsp90) chaperones in mitochondria of human tumor cells triggered compensatory autophagy and an organelle unfolded protein response (UPR) centered on upregulation of CCAAT enhancer binding protein (C/EBP) transcription factors. In turn, this transcriptional UPR repressed NF-κB-dependent gene expression, enhanced tumor cell apoptosis initiated by death receptor ligation, and inhibited intracranial glioblastoma growth in mice without detectable toxicity. These data reveal what we believe to be a novel role of Hsp90 chaperones in the regulation of the protein-folding environment in mitochondria of tumor cells. Disabling this general adaptive pathway could potentially be used in treatment of genetically heterogeneous human tumors.

The type 2 diabetes and insulin-resistance locus near IRS1 is a determinant of HDL cholesterol and triglycerides levels among diabetic subjects.

OBJECTIVE: SNP rs2943641 near the insulin receptor substrate 1 (IRS1) gene has been found to be associated with type 2 diabetes (T2D) and insulin-resistance in genome-wide association studies. We investigated whether this SNP is associated with cardiovascular risk factors and coronary artery disease (CAD) among diabetic individuals. METHODS: SNP rs2943641 was typed in 2133 White T2D subjects and tested for association with BMI, serum HDL cholesterol and triglycerides, hypertension history, and CAD risk. RESULTS: HDL cholesterol decreased by 1mg/dl (p = 0.004) and serum triglycerides increased by 6 mg/dl (p = 0.016) for each copy of the insulin-resistance allele. Despite these effects, no association was found with increased CAD risk (OR = 1.00, 95% CI 0.88-1.13). CONCLUSIONS: The insulin-resistance and T2D locus near the IRS1 gene is a determinant of lower HDL cholesterol among T2D subjects. However, this effect is small and does not translate into a detectable increase in CAD risk in this population.

The ENPP1 Q121 variant predicts major cardiovascular events in high-risk individuals: evidence for interaction with obesity in diabetic patients.

OBJECTIVE: Insulin resistance (IR) and cardiovascular disease may share a common genetic background. We investigated the role of IR-associated ENPP1 K121Q polymorphism (rs1044498) on cardiovascular disease in high-risk individuals. RESEARCH DESIGN AND METHODS: A prospective study (average follow-up, 37 months) was conducted for major cardiovascular events (myocardial infarction [MI], stroke, cardiovascular death) from the Gargano Heart Study (GHS; n = 330 with type 2 diabetes and coronary artery disease), the Tor Vergata Atherosclerosis Study (TVAS; n = 141 who had MI), and the Cardiovascular Risk Extended Evaluation in Dialysis (CREED) database (n = 266 with end-stage renal disease). Age at MI was investigated in cross-sectional studies of 339 type 2 diabetic patients (n = 169 from Italy, n = 170 from the U.S.). RESULTS: Incidence of cardiovascular events per 100 person--years was 4.2 in GHS, 10.8 in TVAS, and 11.7 in CREED. Hazard ratios (HRs) for KQ+QQ versus individuals carrying the K121/K121 genotype (KK) individuals were 1.47 (95% CI 0.80-2.70) in GHS, 2.31 (95% CI 1.22-4.34) in TVAS, and 1.36 (95% CI 0.88-2.10) in CREED, and 1.56 (95% CI 1.15-2.12) in the three cohorts combined. In the 395 diabetic patients, the Q121 variant predicted cardiovascular events among obese but not among nonobese individuals (HR 5.94 vs. 0.62, P = 0.003 for interaction). A similar synergism was observed in cross-sectional studies, with age at MI being 3 years younger in Q121 carriers than in KK homozygotes among obese but not among nonobese patients (P = 0.035 for interaction). CONCLUSIONS: The ENPP1 K121Q polymorphism is an independent predictor of major cardiovascular events in high-risk individuals. In type 2 diabetes, this effect is exacerbated by obesity. Future larger studies are needed to confirm our finding.

Dynein light chain 1 is required for autophagy, protein clearance, and cell death in Drosophila.

Autophagy is a catabolic pathway that is important for turnover of long-lived proteins and organelles, and has been implicated in cell survival, tumor progression, protection from infection, neurodegeneration, and cell death. Autophagy and caspases are required for type II autophagic cell death of Drosophila larval salivary glands during development, but the mechanisms that regulate these degradation pathways are not understood. We conducted a forward genetic screen for genes that are required for salivary gland cell death, and here we describe the identification of Drosophila dynein light chain 1 (ddlc1) as a gene that is required for type II cell death. Autophagy is attenuated in ddlc1 mutants, but caspases are active in these cells. ddlc1 mutant salivary glands develop large fibrillar protein inclusions that stain positive for amyloid-specific dyes and ubiquitin. Ectopic expression of Atg1 is sufficient to induce autophagy, clear protein inclusions, and rescue degradation of ddlc1 mutant salivary glands. Furthermore, ddlc1 mutant larvae have decreased motility, and mutations in ddlc1 enhance the impairment of motility that is observed in a Drosophila model of neurodegenerative disease. Significantly, this decrease in larval motility is associated with decreased clearance of protein with polyglutamine expansion, the accumulation of p62 in neurons and muscles, and fewer synaptic boutons. These results indicate that DDLC1 is required for protein clearance by autophagy that is associated with autophagic cell death and neurodegeneration.

The TRIB3 Q84R polymorphism and risk of early-onset type 2 diabetes.

CONTEXT: The prevalence of type 2 diabetes (T2D), particularly among young adults, has been rising steadily during the past 2 decades. T2D, especially in its early-onset subtype, is under genetic control. TRIB3 inhibits insulin-stimulated Akt phosphorylation and subsequent insulin action. A TRIB3 gain-of-function polymorphism, Q84R (rs2295490), impairs insulin signaling. OBJECTIVE: The objective of the study was to verify the association of TRIB3 Q84R with: 1) T2D, either subtyped or not according to age at diagnosis (early-onset, <45 yr, or >or= 45 yr); 2) insulin secretion and sensitivity in nondiabetic individuals; or 3) in vitro insulin secretion from isolated human islets. DESIGN: Four different case-control samples comprising a total of 5,469 whites were examined. Insulinogenic and insulin sensitivity indexes and their interplay (disposition index) were assessed in 645 nondiabetic individuals at oral glucose tolerance test, glucose (16.7 mmol/liter)-induced in vitro insulin secretion was assessed in islets isolated from 54 nondiabetic donors. RESULTS: In the whole sample, the R84 variant was nominally associated with T2D (odds ratio 1.17, 95% confidence interval 1.00-1.36, P = 0.04). When stratifying according to age of diabetes onset, R84 carriers had an increased risk of early-onset T2D (odds ratio 1.32, 95% confidence interval 1.10-1.58, P = 0.002). Among 645 nondiabetic subjects, R84 carriers had higher glucose levels (P = 0.005) and lower insulinogenic (P = 0.03) and disposition index (P = 0.02) during the oral glucose tolerance test. R84 islets were more likely to display relatively low glucose-stimulated insulin release (P = 0.04). CONCLUSIONS: The TRIB3 R84 variant is associated with early-onset T2D in whites. Alteration in the insulin secretion/insulin sensitivity interplay appears to underlie this association.

A polymorphism at the IL6ST (gp130) locus is associated with traits of the metabolic syndrome.

The interleukin 6 signal transducer (IL6ST, also known as gp130) is a ubiquitously expressed intermediate of the interleukin-6 signaling pathway. We investigated whether genetic variability at the IL6ST locus is involved in the modulation of metabolic traits and the etiology of the metabolic syndrome (MS). Four haplotype-tagging single nucleotide polymorphisms were typed in two populations of non-diabetic subjects, one from Northern Italy (Padua (PD), n = 630), the other from Southern Italy (San Giovanni Rotondo (SGR), n = 553). In the PD population, a nominally significant association was observed between fasting glucose and rs715180 (P = 0.02), rs3729960 (P = 0.02), and rs10940495 (P = 0.05), between homeostasis model assessment index (HOMA(IR)) and rs715180 (P = 0.04), and between triglycerides and rs3729960 (P = 0.03). In the SGR population, high-density lipoprotein (HDL) levels were associated with rs715180 (P = 0.01), systolic blood pressure and waist circumference with rs3729960 (P = 0.005 and 0.02, respectively). The frequency of rs715180 minor allele carriers progressively decreased from individuals with no MS components to those with three or more components (P for trend = 0.006 in the two populations combined). Compared to major allele homozygotes, minor allele carriers had 40% lower odds of having at least one MS component (Odds ratio = 0.6, 95% confidence interval 0.4-0.8, P = 0.005). These findings point to IL6ST variants as possible determinants of impaired glucose metabolism and other abnormalities of MS.

A visfatin promoter polymorphism is associated with low-grade inflammation and type 2 diabetes.

Visfatin (also known as pre-B cell colony-enhancing factor, or PBEF) is a pro-inflammatory adipokine expressed predominantly in visceral fat. We investigated whether polymorphisms at the visfatin/PBEF locus influence the risk of type 2 diabetes (T2D). Linkage disequilibrium analysis of 52 single nucleotide polymorphisms spanning the entire gene (34.7 kb) plus 20.5 kb of the upstream region and 25.5 kb of the downstream region revealed a single haplotype block that could be tagged by seven single nucleotide polymorphisms. These seven tags were typed in a group of T2D patients (n = 814) and a group of non-diabetic controls (n = 320) of white origin. A significant association was observed at -948C>A, with minor allele frequencies of 0.157 in T2D cases and 0.119 in non-diabetic controls (p = 0.021). In a non-diabetic population (n = 630), the same -948 allele that conferred increased risk of T2D was significantly associated with higher plasma levels of fibrinogen and C-reactive protein (p = 0.0022 and 0.0038, respectively). However, no significant associations were observed with BMI, waist circumference, serum glucose levels, or fasting insulin levels. Our findings suggest that the visfatin/PBEF gene may play a role in determining T2D susceptibility, possibly by modulating chronic, low-grade inflammatory responses.

Identification of a locus for maturity-onset diabetes of the young on chromosome 8p23.

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal dominant inheritance and a young onset. Six MODY genes have been discovered to date. To identify additional MODY loci, we conducted a genome scan in 21 extended U.S. families (15 white and 6 from minorities, for a total of 237 individuals) in which MODY was not caused by known MODY genes. Seven chromosomal regions (1q42, 2q24, 2q37, 4p13, 8p23, 11p15, and 19q12) had a parametric heterogeneity logarithm of odds (HLOD) > or =1.00 or a nonparametric logarithm of odds (LOD) > or =0.59 (P < or = 0.05) in the initial screen. After typing additional markers at these loci to reduce the spacing to 2-3 cM, significant linkage was detected on 8p23 (HLOD = 3.37 at D8S1130 and nonparametric LOD = 3.66; P = 2 x 10(-5) at D8S265), where a 4.7-Mb inversion polymorphism is located. Thirty percent of the families (6 of 21) were linked with this region. Another linkage peak on chromosome 2q37 with an HLOD of 1.96 at D2S345/D2S2968 accounted for diabetes in an additional 25% of families (5 of 21). All 6 minority families were among the 11 families linked to these loci. None of the other loci followed up had an HLOD exceeding 1.50. In summary, we have identified a MODY locus on 8p23 that accounts for diabetes in a substantial proportion of MODY cases unlinked to known MODY genes. Another novel MODY locus may be present on 2q37. Cloning these new MODY genes may offer insights to disease pathways that are relevant to the cause of common type 2 diabetes.

Nuclear export of signal recognition particle RNA in mammalian cells.

In mammalian cells the signal recognition particle (SRP) consists of a approximately 300 nucleotide RNA and six proteins. Although the molecular structure and functional cycle of the SRP are both very well understood, far less is known about how the SRP is first assembled in the cell. Recent work has suggested that SRP assembly begins in the nucleoli. When NRK (rat fibroblast) cells were treated with leptomycin B (LMB), a specific inhibitor of the CRM1 nuclear export receptor, the level of SRP RNA increased in the nucleoli, as did the level of nucleolar 28S ribosomal RNA. Moreover, when a hamster cell line carrying a temperature-sensitive mutation in the guanine nucleotide exchange factor of the GTPase Ran (Ran-GEF) was shifted to the non-permissive temperature, the nucleolar level of SRP RNA increased. These results indicate that the steady-state concentration of SRP RNA in the nucleolus is sensitive to perturbations in nuclear import/export pathways.

Genetic modifiers of the age at diagnosis of diabetes (MODY3) in carriers of hepatocyte nuclear factor-1alpha mutations map to chromosomes 5p15, 9q22, and 14q24.

Mutations in hepatocyte nuclear factor (HNF)-1alpha (MODY3) account for the largest proportion of maturity-onset diabetes of the young (MODY) cases in the U.S. This form of diabetes is characterized by impaired insulin secretion in response to glucose, but wide variability exists in the severity of hyperglycemia and in the age at which it becomes clinically manifest. We have previously shown that the age at onset of diabetes in MODY3 families is influenced by familial factors (including modifying genes) and exposure to diabetes in utero. To identify genes influencing the onset of MODY3, we conducted a genome scan in 13 extended MODY families in which diabetes segregates with an HNF-1alpha mutation. Linkage with age at onset of diabetes was assessed by genetic variance component analysis using SOLAR. The locus with the strongest evidence of linkage was on chromosome 14q24 (D14S588; logarithm of odds [LOD] = 2.58, P = 0.0004). This location overlaps with IDDM11 and includes SEL1L, a negative regulator of the Notch pathway that may control islet development. Linkage evidence also supported loci on 5p15 (D5S817; LOD = 2.44, P = 0.0004) and 9q22 (D9S910; LOD = 2.02, P = 0.0018). The latter matches a region linked to 2-h insulin levels in Pima Indians. Less strong linkage evidence was observed at three other regions: chromosomes 3p24 (LOD = 1.44), 7q21 (1.20), and 16q23 (1.51). Our data are consistent with the existence of multiple loci that contribute to the expression of the MODY3 phenotype. Identification of these genes will offer new insights into the pathophysiology of MODY that may, in turn, increase our understanding of the cellular events underlying more common forms of diabetes.