Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5

Poly-ubiquitylation is a common post-translational modification that can impart various functions to a target protein. Several distinct mechanisms have been reported for the assembly of poly-ubiquitin chains, involving either stepwise transfer of ubiquitin monomers or attachment of a preformed poly-ubiquitin chain and requiring either a single pair of ubiquitin-conjugating enzyme (E2) and ubiquitin ligase (E3), or alternatively combinations of different E2s and E3s. We have analysed the mechanism of poly-ubiquitylation of the replication clamp PCNA by two cooperating E2–E3 pairs, Rad6–Rad18 and Ubc13–Mms2–Rad5. We find that the two complexes act sequentially and independently in chain initiation and stepwise elongation, respectively. While loading of PCNA onto DNA is essential for recognition by Rad6–Rad18, chain extension by Ubc13–Mms2–Rad5 is only slightly enhanced by loading. Moreover, in contrast to initiation, chain extension is tolerant to variations in the attachment site of the proximal ubiquitin moiety. Our results provide information about a unique conjugation mechanism that appears to be specialised for a regulatable pattern of dual modification

Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer

Nucleotide sugars are the activated form of monosaccharides used by glycosyltransferases during glycosylation. In eukaryotes the SLC35 family of solute carriers are responsible for their selective uptake into the Endoplasmic Reticulum or Golgi apparatus. The structure of the yeast GDP-mannose transporter, Vrg4, revealed a requirement for short chain lipids and a marked difference in transport rate between the nucleotide sugar and nucleoside monophosphate, suggesting a complex network of regulatory elements control transport into these organelles. Here we report the crystal structure of the GMP bound complex of Vrg4, revealing the molecular basis for GMP recognition and transport. Molecular dynamics, combined with biochemical analysis, reveal a lipid mediated dimer interface and mechanism for coordinating structural rearrangements during transport. Together these results provide further insight into how SLC35 family transporters function within the secretory pathway and sheds light onto the role that membrane lipids play in regulating transport across the membrane

Contributions of ubiquitin- and PCNA-binding domains to the activity of Polymerase η in Saccharomyces cerevisiae

Bypassing of DNA lesions by damage-tolerant DNA polymerases depends on the interaction of these enzymes with the monoubiquitylated form of the replicative clamp protein, PCNA. We have analyzed the contributions of ubiquitin and PCNA binding to damage bypass and damage-induced mutagenesis in Polymerase η (encoded by RAD30) from the budding yeast Saccharomyces cerevisiae. We report here that a ubiquitin-binding domain provides enhanced affinity for the ubiquitylated form of PCNA and is essential for in vivo function of the polymerase, but only in conjunction with a basal affinity for the unmodified clamp, mediated by a conserved PCNA interaction motif. We show that enhancement of the interaction and function in damage tolerance does not depend on the ubiquitin attachment site within PCNA. Like its mammalian homolog, budding yeast Polymerase η itself is ubiquitylated in a manner dependent on its ubiquitin-binding domain

MemProtMD: Automated Insertion of Membrane Protein Structures into Explicit Lipid Membranes

SummaryThere has been exponential growth in the number of membrane protein structures determined. Nevertheless, these structures are usually resolved in the absence of their lipid environment. Coarse-grained molecular dynamics (CGMD) simulations enable insertion of membrane proteins into explicit models of lipid bilayers. We have automated the CGMD methodology, enabling membrane protein structures to be identified upon their release into the PDB and embedded into a membrane. The simulations are analyzed for protein-lipid interactions, identifying lipid binding sites, and revealing local bilayer deformations plus molecular access pathways within the membrane. The coarse-grained models of membrane protein/bilayer complexes are transformed to atomistic resolution for further analysis and simulation. Using this automated simulation pipeline, we have analyzed a number of recently determined membrane protein structures to predict their locations within a membrane, their lipid/protein interactions, and the functional implications of an enhanced understanding of the local membrane environment of each protein

Biological Variation of Plasma and Urinary Markers of Acute Kidney Injury in Patients with Chronic Kidney Disease

BACKGROUND: Identification of acute kidney injury (AKI) is predominantly based on changes in plasma creatinine concentration, an insensitive marker. Alternative biomarkers have been proposed. The reference change value (RCV), the point at which biomarker change can be inferred to have occurred with statistical certainty, provides an objective assessment of change in serial tests results in an individual. METHODS: In 80 patients with chronic kidney disease, weekly measurements of blood and urinary biomarker concentrations were undertaken over 6 weeks. Variability was determined and compared before and after adjustment for urinary creatinine and across subgroups stratified by level of kidney function, proteinuria, and presence or absence of diabetes. RESULTS: RCVs were determined for whole blood, plasma, and urinary neutrophil gelatinase-associated lipocalin (111%, 59%, and 693%, respectively), plasma cystatin C (14%), creatinine (17%), and urinary kidney injury molecule 1 (497%), tissue inhibitor of metalloproteinases 2 (454%), N-acetyl-?-d-glucosaminidase (361%), interleukin-18 (819%), albumin (430%), and ?1-microglobulin (216%). Blood biomarkers exhibited lower variability than urinary biomarkers. Generally, adjusting urinary biomarker concentrations for creatinine reduced (P < 0.05) within-individual biological variability (CVI). For some markers, variation differed (P < 0.05) between subgroups. CONCLUSIONS: These data can form a basis for application of these tests in clinical practice and research studies and are applicable across different levels of kidney function and proteinuria and in the presence or absence of diabetes. Most of the studied biomarkers have relatively high CVI (noise) but also have reported large concentration changes in response to renal insult (signal); thus progressive change should be detectable (high signal-to-noise ratio) when baseline data are available

Outcomes in patients with gunshot wounds to the brain.

Introduction:Gunshot wounds to the brain (GSWB) confer high lethality and uncertain recovery. It is unclear which patients benefit from aggressive resuscitation, and furthermore whether patients with GSWB undergoing cardiopulmonary resuscitation (CPR) have potential for survival or organ donation. Therefore, we sought to determine the rates of survival and organ donation, as well as identify factors associated with both outcomes in patients with GSWB undergoing CPR. Methods:We performed a retrospective, multicenter study at 25 US trauma centers including dates between June 1, 2011 and December 31, 2017. Patients were included if they suffered isolated GSWB and required CPR at a referring hospital, in the field, or in the trauma resuscitation room. Patients were excluded for significant torso or extremity injuries, or if pregnant. Binomial regression models were used to determine predictors of survival/organ donation. Results:825 patients met study criteria; the majority were male (87.6%) with a mean age of 36.5 years. Most (67%) underwent CPR in the field and 2.1% (n=17) survived to discharge. Of the non-survivors, 17.5% (n=141) were considered eligible donors, with a donation rate of 58.9% (n=83) in this group. Regression models found several predictors of survival. Hormone replacement was predictive of both survival and organ donation. Conclusion:We found that GSWB requiring CPR during trauma resuscitation was associated with a 2.1% survival rate and overall organ donation rate of 10.3%. Several factors appear to be favorably associated with survival, although predictions are uncertain due to the low number of survivors in this patient population. Hormone replacement was predictive of both survival and organ donation. These results are a starting point for determining appropriate treatment algorithms for this devastating clinical condition. Level of evidence:Level II

On the temperature dependence of enzyme-catalyzed rates

One of the critical variables that determine the rate of any reaction is temperature. For biological systems, the effects of temperature are convoluted with myriad (and often opposing) contributions from enzyme catalysis, protein stability, and temperature-dependent regulation, for example. We have coined the phrase “macromolecular rate theory (MMRT)” to describe the temperature dependence of enzyme-catalyzed rates independent of stability or regulatory processes. Central to MMRT is the observation that enzyme-catalyzed reactions occur with significant values of Δ<i>C</i>_<i>p</i>^‡ that are in general negative. That is, the heat capacity (<i>C</i>_<i>p</i>) for the enzyme–substrate complex is generally larger than the <i>C</i>_<i>p</i> for the enzyme-transition state complex. Consistent with a classical description of enzyme catalysis, a negative value for Δ<i>C</i>_<i>p</i>^‡ is the result of the enzyme binding relatively weakly to the substrate and very tightly to the transition state. This observation of negative Δ<i>C</i>_<i>p</i>^‡ has important implications for the temperature dependence of enzyme-catalyzed rates. Here, we lay out the fundamentals of MMRT. We present a number of hypotheses that arise directly from MMRT including a theoretical justification for the large size of enzymes and the basis for their optimum temperatures. We rationalize the behavior of psychrophilic enzymes and describe a “psychrophilic trap” which places limits on the evolution of enzymes in low temperature environments. One of the defining characteristics of biology is catalysis of chemical reactions by enzymes, and enzymes drive much of metabolism. Therefore, we also expect to see characteristics of MMRT at the level of cells, whole organisms, and even ecosystems

Estimation of changes in the force of infection for intestinal and urogenital schistosomiasis in countries with Schistosomiasis Control Initiative-assisted programmes

The last decade has seen an expansion of national schistosomiasis control programmes in Africa based on large-scale preventative chemotherapy. In many areas this has resulted in considerable reductions in infection and morbidity levels in treated individuals. In this paper, we quantify changes in the force of infection (FOI), defined here as the per (human) host parasite establishment rate, to ascertain the impact on transmission of some of these programmes under the umbrella of the Schistosomiasis Control Initiative (SCI)

Diagnostic and treatment pathways for men with prostate cancer in Queensland: investigating spatial and demographic inequalities

Background: Patterns of diagnosis and management for men diagnosed with prostate cancer in Queensland, Australia, have not yet been systematically documented and so assumptions of equity are untested. This longitudinal study investigates the association between prostate cancer diagnostic and treatment outcomes and key area-level characteristics and individual-level demographic, clinical and psychosocial factors.---------- Methods/Design: A total of 1064 men diagnosed with prostate cancer between February 2005 and July 2007 were recruited through hospital-based urology outpatient clinics and private practices in the centres of Brisbane, Townsville and Mackay (82% of those referred). Additional clinical and diagnostic information for all 6609 men diagnosed with prostate cancer in Queensland during the study period was obtained via the population-based Queensland Cancer Registry. Respondent data are collected using telephone and self-administered questionnaires at pre-treatment and at 2 months, 6 months, 12 months, 24 months, 36 months, 48 months and 60 months post-treatment. Assessments include demographics, medical history, patterns of care, disease and treatment characteristics together with outcomes associated with prostate cancer, as well as information about quality of life and psychological adjustment. Complementary detailed treatment information is abstracted from participants’ medical records held in hospitals and private treatment facilities and collated with health service utilisation data obtained from Medicare Australia. Information about the characteristics of geographical areas is being obtained from data custodians such as the Australian Bureau of Statistics. Geo-coding and spatial technology will be used to calculate road travel distances from patients’ residences to treatment centres. Analyses will be conducted using standard statistical methods along with multilevel regression models including individual and area-level components.---------- Conclusions: Information about the diagnostic and treatment patterns of men diagnosed with prostate cancer is crucial for rational planning and development of health delivery and supportive care services to ensure equitable access to health services, regardless of geographical location and individual characteristics. This study is a secondary outcome of the randomised controlled trial registered with the Australian New Zealand Clinical Trials Registry (ACTRN12607000233426

Parent-of-origin-specific allelic associations among 106 genomic loci for age at menarche.

Age at menarche is a marker of timing of puberty in females. It varies widely between individuals, is a heritable trait and is associated with risks for obesity, type 2 diabetes, cardiovascular disease, breast cancer and all-cause mortality. Studies of rare human disorders of puberty and animal models point to a complex hypothalamic-pituitary-hormonal regulation, but the mechanisms that determine pubertal timing and underlie its links to disease risk remain unclear. Here, using genome-wide and custom-genotyping arrays in up to 182,416 women of European descent from 57 studies, we found robust evidence (P < 5 × 10(-8)) for 123 signals at 106 genomic loci associated with age at menarche. Many loci were associated with other pubertal traits in both sexes, and there was substantial overlap with genes implicated in body mass index and various diseases, including rare disorders of puberty. Menarche signals were enriched in imprinted regions, with three loci (DLK1-WDR25, MKRN3-MAGEL2 and KCNK9) demonstrating parent-of-origin-specific associations concordant with known parental expression patterns. Pathway analyses implicated nuclear hormone receptors, particularly retinoic acid and γ-aminobutyric acid-B2 receptor signalling, among novel mechanisms that regulate pubertal timing in humans. Our findings suggest a genetic architecture involving at least hundreds of common variants in the coordinated timing of the pubertal transition