The San1 Ubiquitin Ligase Avidly Recognizes Misfolded Proteins Through Multiple Substrate Binding Sites

Cellular homeostasis depends on robust protein quality control (PQC) pathways that discern misfolded proteins from functional ones in the cell. One major branch of PQC involves the controlled degradation of misfolded proteins by the ubiquitin-proteasome system. Here ubiquitin ligases must recognize and bind to misfolded proteins with sufficient energy to form a complex and with an adequate half-life to achieve poly-ubiquitin chain formation, the signal for protein degradation, prior to its dissociation from the ligase. It is not well understood how PQC ubiquitin ligases accomplish these tasks. Employing a fully reconstituted enzyme and substrate system to perform quantitative biochemical experiments, we demonstrate that the yeast PQC ubiquitin ligase San1 contains multiple substrate binding sites along its polypeptide chain that appear to display specificity for unique misfolded proteins. The results are consistent with a model where these substrate binding sites enable San1 to bind to misfolded substrates avidly, resulting in high affinity ubiquitin ligase-substrate complexes

SUDDEN CARDIAC DEATH: ROLE OF LEFT VENTRICULAR DYSFUNCTION

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72037/1/j.1749-6632.1982.tb55219.x.pd

Phosfinder: a web server for the identification of phosphate-binding sites on protein structures

Phosfinder is a web server for the identification of phosphate binding sites in protein structures. Phosfinder uses a structural comparison algorithm to scan a query structure against a set of known 3D phosphate binding motifs. Whenever a structural similarity between the query protein and a phosphate binding motif is detected, the phosphate bound by the known motif is added to the protein structure thus representing a putative phosphate binding site. Predicted binding sites are then evaluated according to (i) their position with respect to the query protein solvent-excluded surface and (ii) the conservation of the binding residues in the protein family. The server accepts as input either the PDB code of the protein to be analyzed or a user-submitted structure in PDB format. All the search parameters are user modifiable. Phosfinder outputs a list of predicted binding sites with detailed information about their structural similarity with known phosphate binding motifs, and the conservation of the residues involved. A graphical applet allows the user to visualize the predicted binding sites on the query protein structure. The results on a set of 52 apo/holo structure pairs show that the performance of our method is largely unaffected by ligand-induced conformational changes. Phosfinder is available at http://phosfinder.bio.uniroma2.it

Ubiquitin ligation to F-box protein targets by SCF-RBR E3-E3 super-assembly

E3 ligases are typically classified by hallmark domains such as RING and RBR, which are thought to specify unique catalytic mechanisms of ubiquitin transfer to recruited substrates(1,2). However, rather than functioning individually, many neddylated cullin-RING E3 ligases (CRLs) and RBR-type E3 ligases in the ARIH family-which together account for nearly half of all ubiquitin ligases in humans-form E3-E3 super-assemblies(3-7). Here, by studying CRLs in the SKP1-CUL1-F-box (SCF) family, we show how neddylated SCF ligases and ARIH1 (an RBR-type E3 ligase) co-evolved to ubiquitylate diverse substrates presented on various F-box proteins. We developed activity-based chemical probes that enabled cryo-electron microscopy visualization of steps in E3-E3 ubiquitylation, initiating with ubiquitin linked to the E2 enzyme UBE2L3, then transferred to the catalytic cysteine of ARIH1, and culminating in ubiquitin linkage to a substrate bound to the SCF E3 ligase. The E3-E3 mechanism places the ubiquitin-linked active site of ARIH1 adjacent to substrates bound to F-box proteins (for example, substrates with folded structures or limited length) that are incompatible with previously described conventional RING E3-only mechanisms. The versatile E3-E3 super-assembly may therefore underlie widespread ubiquitylation.Chemical Immunolog

Antecedent Hypoglycemia Impairs Autonomic Cardiovascular Function: Implications for Rigorous Glycemic Control

OBJECTIVE— Glycemic control decreases the incidence and progression of diabetic complications but increases the incidence of hypoglycemia. Hypoglycemia can impair hormonal and autonomic responses to subsequent hypoglycemia. Intensive glycemic control may increase mortality in individuals with type 2 diabetes at high risk for cardiovascular complications. We tested the hypothesis that prior exposure to hypoglycemia leads to impaired cardiovascular autonomic function

Reproducibility of Heart Rate Variability Indices in Children with Cystic Fibrosis

Fundamental to the potential utilisation of heart rate variability (HRV) indices as a prognostic tool is the reproducibility of these measures. The purpose of the present study was therefore to investigate the reproducibility of 24-hour derived HRV indices in a clinical paediatric population. Eighteen children (10 boys; 12.4 ± 2.8 years) with mild to moderate Cystic Fibrosis (CF; FVC: 83 ± 12% predicted; FEV1: 80 ± 9% predicted) and eighteen age- and sex-matched controls (10 boys; 12.5 ± 2.7 years) wore a combined ECG and accelerometer for two consecutive days. Standard time and frequency domain indices of HRV were subsequently derived. Reproducibility was assessed by Bland-Altman plots, 95% limits of agreement and intra-class correlation coefficients (ICC). In both groups, there was no systematic difference between days, with the variables demonstrating a symmetrical, homoscedastic distribution around the zero line. The time domain parameters demonstrated a good to excellent reproducibility irrespective of the population considered (ICC: 0.56 to 0.86). In contrast, whilst the frequency domain parameters similarly showed excellent reproducibility in the healthy children (ICC: 0.70 to 0.96), the majority of the frequency domain parameters illustrated a poor to moderate reproducibility in those with CF (ICC: 0.22 to 0.43). The exceptions to this trend were the normalised LF and HF components which were associated with a good to excellent reproducibility. These findings thereby support the utilisation of time and relative frequency domain HRV indices as a prognostic tool in children with CF. Furthermore, the present results highlight the excellent reproducibility of HRV in healthy children, indicating that this may be a useful tool to assess intervention effectiveness in this population

Mutations at the flavin binding site of ETF:QO yield a MADD-like severe phenotype in Drosophila

Following a screening on EMS-induced Drosophila mutants defective for formation and morphogenesis of epithelial cells, we have identified three lethal mutants defective for the production of embryonic cuticle. The mutants are allelic to the CG12140 gene, the fly homologue of electron transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). In humans, inherited defects in this inner membrane protein account for multiple acyl-CoA dehydrogenase deficiency (MADD), a metabolic disease of beta-oxidation, with a broad range of clinical phenotypes, varying from embryonic lethal to mild forms. The three mutant alleles carried distinct missense mutations in ETF:QO (G65E, A68V and S104F) and maternal mutant embryos for ETF:QO showed lethal morphogenetic defects and a significant induction of apoptosis following germ-band elongation. This phenotype is accompanied by an embryonic accumulation of short- and medium-chain acylcarnitines (C4. C8 and 02) as well as long-chain acylcarnitines (C14 and C16:1), whose elevation is also found in severe MADD forms in humans under intense metabolic decompensation. In agreement the ETF:QO activity in the mutant embryos is markedly decreased in relation to wild type activity. Amino acid sequence analysis and structural mapping into a molecular model of ETF:QO show that all mutations map at FAD interacting residues, two of which at the nucleotide-binding Rossmann fold. This structural domain is composed by a beta-strand connected by a short loop to an alpha-helix, and its perturbation results in impaired cofactor association via structural destabilisation and consequently enzymatic inactivation. This work thus pinpoints the molecular origins of a severe MADD-like phenotype in the fruit fly and establishes the proof of concept concerning the suitability of this organism as,a potential model organism for MADD. (C) 2012 Elsevier B.V. All rights reserved.Fundacao para a Ciencia e Tecnologia (FCT/MCTES, Portugal) [PTDC/SAU-GMG/70033/2006, PTDC/QUI-BIQ/113027/2009, PTDC/BIA-BCM/111822/2009, PTDC/SAU-BID/111796/2009, SFRH/BPD/41609/2007, SFRH/BPD/74475/2010, SFRH/BPD/34763/2007]; CLIMB UK; [PEst-OE/EQB/LA0004/2011]info:eu-repo/semantics/publishedVersio

The distal fascicle of the anterior inferior tibiofibular ligament as a cause of tibiotalar impingement syndrome: a current concepts review

Impingement syndromes of the ankle involve either osseous or soft tissue impingement and can be anterior, anterolateral, or posterior. Ankle impingement syndromes are painful conditions caused by the friction of joint tissues, which are both the cause and the effect of altered joint biomechanics. The distal fascicle of the anterior inferior tibiofibular ligament (AITFL) is possible cause of anterior impingement. The objective of this article was to review the literature concerning the anatomy, pathogenesis, symptoms and treatment of the AITFL impingement and finally to formulate treatment recommendations. The AITFL starts from the distal tibia, 5 mm in average above the articular surface, and descends obliquely between the adjacent margins of the tibia and fibula, anterior to the syndesmosis to the anterior aspect of the lateral malleolus. The incidence of the accessory fascicle differs very widely in the several studies. The presence of the distal fascicle of the AITFL and also the contact with the anterolateral talus is probably a normal finding. It may become pathological, due to anatomical variations and/or anterolateral instability of the ankle resulting from an anterior talofibular ligament injury. When observed during an ankle arthroscopy, the surgeon should look for the criteria described to decide whether it is pathological and considering resection of the distal fascicle. The presence of the AITFL and the contact with the talus is a normal finding. An impingement of the AITFL can result from an anatomical variant or anteroposterior instability of the ankle. The diagnosis of ligamentous impingement in the anterior aspect of the ankle should be considered in patients who have chronic ankle pain in the anterolateral aspect of the ankle after an inversion injury and have a stable ankle, normal plain radiographs, and isolated point tenderness on the anterolateral aspect of the talar dome and in the anteroinferior tibiofibular ligament. The impingement syndrome can be treated arthroscopically

Chaotic Signatures of Heart Rate Variability and Its Power Spectrum in Health, Aging and Heart Failure

A paradox regarding the classic power spectral analysis of heart rate variability (HRV) is whether the characteristic high- (HF) and low-frequency (LF) spectral peaks represent stochastic or chaotic phenomena. Resolution of this fundamental issue is key to unraveling the mechanisms of HRV, which is critical to its proper use as a noninvasive marker for cardiac mortality risk assessment and stratification in congestive heart failure (CHF) and other cardiac dysfunctions. However, conventional techniques of nonlinear time series analysis generally lack sufficient sensitivity, specificity and robustness to discriminate chaos from random noise, much less quantify the chaos level. Here, we apply a ‘litmus test’ for heartbeat chaos based on a novel noise titration assay which affords a robust, specific, time-resolved and quantitative measure of the relative chaos level. Noise titration of running short-segment Holter tachograms from healthy subjects revealed circadian-dependent (or sleep/wake-dependent) heartbeat chaos that was linked to the HF component (respiratory sinus arrhythmia). The relative ‘HF chaos’ levels were similar in young and elderly subjects despite proportional age-related decreases in HF and LF power. In contrast, the near-regular heartbeat in CHF patients was primarily nonchaotic except punctuated by undetected ectopic beats and other abnormal beats, causing transient chaos. Such profound circadian-, age- and CHF-dependent changes in the chaotic and spectral characteristics of HRV were accompanied by little changes in approximate entropy, a measure of signal irregularity. The salient chaotic signatures of HRV in these subject groups reveal distinct autonomic, cardiac, respiratory and circadian/sleep-wake mechanisms that distinguish health and aging from CHF

Pathogenic Bacteria Target NEDD8-Conjugated Cullins to Hijack Host-Cell Signaling Pathways

The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G1/S and G2/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21waf1 and p27kip1. Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria