The covalent attachment of ubiquitin (Ub) to short-lived or damaged proteins is believed to be the signal that initiates their selective degradation. In several cases, it has been shown that the proteolytic signal takes the form of a multi-Ub chain in which successive Ub molecules are linked tandemly at lysine 48 (K-48). Here we show that Ub molecules can be linked together in vivo at two other lysine positions, lysine 29 (K-29) and lysine 63 (K-63). The formation of these alternative linkages is strongly dependent on the presence of the stress-related Ub conjugating enzymes UBC4 and UBC5. Furthermore, expression of Ub carrying a K-63 to arginine 63 substitution in a strain of Saccharomyces cerevisiae that is missing the poly-Ub gene, UBI4, fails to compensate for the stress defects associated with these cells. Taken together, these results suggest that the formation of multi-Ub chains involving K-63 linkages plays an important role in the yeast stress response. In broader terms, these results also suggest that Ub is a versatile signal in which different Ub chain configurations are used for different functions.

AIMTo determine the short-term biochemical effects and clinical tolerability of intermittent fasting (IF) in adults with type 2 diabetes mellitus (T2DM).METHODSWe describe a three-phase observational study (baseline 2 wk, intervention 2 wk, follow-up 2 wk) designed to determine the clinical, biochemical, and tolerability of IF in community-dwelling volunteer adults with T2DM. Biochemical, anthropometric, and physical activity measurements (using the Yale Physical Activity Survey) were taken at the end of each phase. Participants reported morning, afternoon and evening self-monitored blood glucose (SMBG) and fasting duration on a daily basis throughout all study stages, in addition to completing a remote food photography diary three times within each study phase. Fasting blood samples were collected on the final days of each study phase.RESULTSAt baseline, the ten participants had a confirmed diagnosis of T2DM and were all taking metformin, and on average were obese [mean body mass index (BMI) 36.90 kg/m2]. We report here that a short-term period of IF in a small group of individuals with T2DM led to significant group decreases in weight (-1.395 kg, P = 0.009), BMI (-0.517, P = 0.013), and at-target morning glucose (SMBG). Although not a study requirement, all participants preferentially chose eating hours starting in the midafternoon. There was a significant increase (P < 0.001) in daily hours fasted in the IF phase (+5.22 h), although few attained the 18-20 h fasting goal (mean 16.82 ± 1.18). The increased fasting duration improved at-goal (< 7.0 mmol/L) morning SMBG to 34.1%, from a baseline of 13.8%. Ordinal Logistic Regression models revealed a positive relationship between the increase in hours fasted and fasting glucose reaching target values (χ2 likelihood ratio = 8.36, P = 0.004) but not for afternoon or evening SMBG (all P > 0.1). Postprandial SMBGs were also improved during the IF phase, with 60.5% readings below 9.05 mmol/L, compared to 52.6% at baseline, and with less glucose variation. Neither insulin resistance (HOMA-IR), nor inflammatory markers (C-reactive protein) normalized during the IF phase. IF led to an overall spontaneous decrease in caloric intake as measured by food photography (Remote Food Photography Method). The data demonstrated discernable trends during IF for lower energy, carbohydrate, and fat intake when compared to baseline. Physical activity, collected by a standardized measurement tool (Yale Physical Activity Survey), increased during the intervention phase and subsequently decreased in the follow-up phase. IF was well tolerated in the majority of individuals with 6/10 participants stating they would continue with the IF regimen after the completion of the study, in a full or modified capacity (i.e., every other day or reduced fasting hours).CONCLUSIONThe results from this pilot study indicate that short-term daily IF may be a safe, tolerable, dietary intervention in T2DM patients that may improve key outcomes including body weight, fasting glucose and postprandial variability....

The UBC1 ubiquitin-conjugating enzyme from Saccharomyces cerevisiae has an overlapping function with the UBC4 and UBC5 enzymes in the yeast stress response and an important role in the G0 to G1 transition that accompanies spore germination (Seufert, W., McGrath, J. P., and Jentsch, S. (1990) EMBO J. 9, 4573-4541). In the present work we report that the UBC1 enzyme assembles onto itself a multi-ubiquitin chain in vitro whose linkage configuration is dependent on the unconserved carboxyl-terminal extension or tail that is appended to its catalytic domain. Using chemical cleavage and site-specific mutagenesis, we have mapped the location of the chain to lysine 93 which lies near the active site within the catalytic domain. The ubiquitin molecule that anchors the chain is transferred to this lysine from the active site of the same UBC1 molecule. When the tail of UBC1 is deleted, the catalytic domain synthesizes a chain that consists of ubiquitin molecules uniformly linked to one another via lysine 48. In the presence of the tail, however, a chain is assembled that is composed of linkages that are stable to alkali but which do not utilize lysines. Furthermore, when the amino terminus of ubiquitin is blocked by an appended peptide tag, chain assembly reverts from this alternative configuration to the canonical lysine 48 variety. Taken together, these results suggest that the alternative chain is composed of linkages in which one ubiquitin molecule forms a peptide bond with the alpha-amino terminus of another, thereby supporting the emerging view that Ub can be attached to itself or other proteins in a variety of ways.

It is well recognized that bone loss accelerates in hypogonadal states, with female menopause being the classic example of sex hormones affecting the regulation of bone metabolism. Underrepresented is our knowledge of the clinical and metabolic consequences of overt male hypogonadism, as well as the more subtle age-related decline in testosterone on bone quality. While menopause and estrogen deficiency are well-known risk factors for osteoporosis in women, the effects of age-related testosterone decline in men on bone health are less well known. Much of our knowledge comes from observational studies and retrospective analysis on small groups of men with variable causes of primary or secondary hypogonadism and mild to overt testosterone deficiencies. This review aims to present the current knowledge of the consequences of adult male hypogonadism on bone metabolism. The direct and indirect effects of testosterone on bone cells will be explored as well as the important differences in male osteoporosis and assessment as compared to that in females. The clinical consequence of both primary and secondary hypogonadism, as well as testosterone decline in older males, on bone density and fracture risk in men will be summarized. Finally, the therapeutic options and their efficacy in male osteoporosis and hypogonadism will be discussed.

Multiple drug resistant (MDR) malignancy remains a predictable and often terminal event in cancer therapy, and affects individuals with many cancer types, regardless of the stage at which they were originally diagnosed or the interval from last treatment. Protein biomarkers of MDR are not globally used for clinical decision-making, but include the overexpression of drug-efflux pumps (ABC transporter family) such as MDR-1 and BCRP, as well as HIF1α, a stress responsive transcription factor found elevated within many MDR tumors. Here, we present the important in vitro discovery that the development of MDR (in breast cancer cells) can be prevented, and that established MDR could be resensitized to therapy, by adjunct treatment with metformin. Metformin is prescribed globally to improve insulin sensitivity, including in those individuals with Type 2 Diabetes Mellitus (DM2). We demonstrate the effectiveness of metformin in resensitizing MDR breast cancer cell lines to their original treatment, and provide evidence that metformin may function through a mechanism involving post-translational histone modifications via an indirect histone deacetylase inhibitor (HDACi) activity. We find that metformin, at low physiological concentrations, reduces the expression of multiple classic protein markers of MDR in vitro and in preliminary in vivo models. Our demonstration that metformin can prevent MDR development and resensitize MDR cells to chemotherapy in vitro, provides important medical relevance towards metformin’s potential clinical use against MDR cancers.