Regulation of peptide import through phosphorylation of Ubr1, the ubiquitin ligase of the N-end rule pathway

Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. These residues are recognized by E3 ubiquitin ligases called N-recognins. Ubr1 is the N-recognin of the yeast Saccharomyces cerevisiae. Extracellular amino acids or short peptides up-regulate the peptide transporter gene PTR2, thereby increasing the capacity of a cell to import peptides. Cup9 is a transcriptional repressor that down-regulates PTR2. The induction of PTR2 by peptides or amino acids involves accelerated degradation of Cup9 by the N-end rule pathway. We report here that the Ubr1 N-recognin, which conditionally targets Cup9 for degradation, is phosphorylated in vivo at multiple sites, including Ser300 and Tyr277. We also show that the type-I casein kinases Yck1 and Yck2 phosphorylate Ubr1 on Ser300, and thereby make possible (“prime”) the subsequent (presumably sequential) phosphorylations of Ubr1 on Ser296, Ser292, Thr288, and Tyr277 by Mck1, a kinase of the glycogen synthase kinase 3 (Gsk3) family. Phosphorylation of Ubr1 on Tyr277 by Mck1 is a previously undescribed example of a cascade-based tyrosine phosphorylation by a Gsk3-type kinase outside of autophosphorylation. We show that the Yck1/Yck2-mediated phosphorylation of Ubr1 on Ser300 plays a major role in the control of peptide import by the N-end rule pathway. In contrast to phosphorylation on Ser300, the subsequent (primed) phosphorylations, including the one on Tyr277, have at most minor effects on the known properties of Ubr1, including regulation of peptide import. Thus, a biological role of the rest of Ubr1 phosphorylation cascade remains to be identified

Chew-Bites, Jaw Movement Allocation and Bite Rate in Grazing Cattle as Identified by Acoustic Monitoring

Bite rate derives from the time budget of the biting and chewing processes of intake, which are both performed by jaw movements. A new type of jaw movement was revealed by acoustic monitoring in cattle - the chew-bite -which chews herbage already in the mouth and harvests fresh herbage with the same jaw movement (Laca et al., 1992). Chew-biting should enable the animal to reduce the total number of jaw movements performed per bite without reducing the number of chews per bite. We examined the variation among individuals in the allocation of jaw movements between the three types, and its relation to bite rate

The Importance of Patch Size in Estimating Steady-State Bite Rate in Grazing Cattle

Since the pioneering work of Black and Kenney (1984), various intake studies have been conducted at the spatial scale of a single feeding station ( patch ) to elucidate the processes that determine instantaneous intake rate (e.g. Laca et al., 1994). While these are well-suited for patch depletion studies, it is less clear how well they represent non-patchy and relatively homogeneous environments (Ungar & Griffiths, 2002). Clearly, grazing should be restricted to the upper grazing horizon (i.e. layer of bites), but sample duration may be insufficient to characterize steady-state behaviour, especially when grazing commences on an empty mouth. We examined the impact of feeding station size on bite rate and jaw movement allocation between bites and chews

Tumour micro-environment elicits innate resistance to RAF inhibitors through HGF secretion.

Drug resistance presents a challenge to the treatment of cancer patients. Many studies have focused on cell-autonomous mechanisms of drug resistance. By contrast, we proposed that the tumour micro-environment confers innate resistance to therapy. Here we developed a co-culture system to systematically assay the ability of 23 stromal cell types to influence the innate resistance of 45 cancer cell lines to 35 anticancer drugs. We found that stroma-mediated resistance is common, particularly to targeted agents. We characterized further the stroma-mediated resistance of BRAF-mutant melanoma to RAF inhibitors because most patients with this type of cancer show some degree of innate resistance. Proteomic analysis showed that stromal cell secretion of hepatocyte growth factor (HGF) resulted in activation of the HGF receptor MET, reactivation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI(3)K)-AKT signalling pathways, and immediate resistance to RAF inhibition. Immunohistochemistry experiments confirmed stromal cell expression of HGF in patients with BRAF-mutant melanoma and showed a significant correlation between HGF expression by stromal cells and innate resistance to RAF inhibitor treatment. Dual inhibition of RAF and either HGF or MET resulted in reversal of drug resistance, suggesting RAF plus HGF or MET inhibitory combination therapy as a potential therapeutic strategy for BRAF-mutant melanoma. A similar resistance mechanism was uncovered in a subset of BRAF-mutant colorectal and glioblastoma cell lines. More generally, this study indicates that the systematic dissection of interactions between tumours and their micro-environment can uncover important mechanisms underlying drug resistance

Dupuytren's disease in bosnia and herzegovina. An epidemiological study

BACKGROUND: It is generally held that Dupuytren's disease is more common in northern than in southern Europe, but there are very few studies from southern European countries. METHODS: We examined the hands of 1207 men and women over the age of 50 years in Bosnia and Herzegovina. RESULTS: The prevalence of Dupuytren's disease was highly age-dependent, ranging from 17% for men between 50–59 years to 60% in the oldest men. The prevalence among women was lower. The great majority only had palmar changes without contracture of the digit. The prevalence was significantly lower among Bosnian Muslim men than among Bosnian Croat and Serbian men and significantly increased among diabetics. No association could be detected between Dupuytren's disease and smoking, alcohol consumption or living in rural or urban areas. CONCLUSION: We conclude that, contrary to previous opinion, Dupuytren's disease is common in Bosnia and Herzegovina

Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase

O^6-methylguanine (O^6meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. O^6meG is repaired through its demethylation by the O^6-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated O^6 in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system

Unanswered ethical and scientific questions for trials of invasive interventions for coronary disease: The case of single vessel disease

Trials in the 1990s demonstrated that medical therapy is as effective as invasive therapies for treating single-vessel coronary disease. Yet more recent studies enrolling patients with this condition have focused on evaluating only invasive approaches, namely, stenting versus coronary artery bypass surgery. Several ethical and scientific questions remain unanswered regarding the conduct of these later trials. Were they justified? Why wasn't a medical therapy arm included? Were subjects informed about the availability of medical therapy as an equivalent option? Was optimized medical therapy given prior to randomization? The absence of clear answers to these questions raises the possibility of serious bias in favor of invasive interventions. Considering that medical therapy is underutilized in patients with coronary disease, efforts should focus more on increasing utilization of medical therapy and proper selection of noninvasive interventions

Reno-protective effects of renin–angiotensin system blockade in type 2 diabetic patients: a systematic review and network meta-analysis

AIMS/HYPOTHESIS: This meta-analysis aimed to compare the renal outcomes between ACE inhibitor (ACEI)/angiotensin II receptor blocker (ARB) and other antihypertensive drugs or placebo in type 2 diabetes. METHODS: Publications were identified from Medline and Embase up to July 2011. Only randomised controlled trials comparing ACEI/ARB monotherapy with other active drugs or placebo were eligible. The outcome of end-stage renal disease, doubling of serum creatinine, microvascular complications, microalbuminuria, macroalbuminuria and albuminuria regression were extracted. Risk ratios were pooled using a random-effects model if heterogeneity was present; a fixed-effects model was used in the absence of heterogeneity. RESULTS: Of 673 studies identified, 28 were eligible (n = 13-4,912). In direct meta-analysis, ACEI/ARB had significantly lower risk of serum creatinine doubling (pooled RR = 0.66 [95% CI 0.52, 0.83]), macroalbuminuria (pooled RR = 0.70 [95% CI 0.50, 1.00]) and albuminuria regression (pooled RR 1.16 [95% CI 1.00, 1.39]) than other antihypertensive drugs, mainly calcium channel blockers (CCBs). Although the risks of end-stage renal disease and microalbuminuria were lower in the ACEI/ARB group (pooled RR 0.82 [95% CI 0.64, 1.05] and 0.84 [95% CI 0.61, 1.15], respectively), the differences were not statistically significant. The ACEI/ARB benefit over placebo was significant for all outcomes except microalbuminuria. A network meta-analysis detected significant treatment effects across all outcomes for both active drugs and placebo comparisons. CONCLUSIONS/INTERPRETATION: Our review suggests a consistent reno-protective effect of ACEI/ARB over other antihypertensive drugs, mainly CCBs, and placebo in type 2 diabetes. The lack of any differences in BP decrease between ACEI/ARB and active comparators suggest this benefit is not due simply to the antihypertensive effect