Proteomic techniques to probe the ubiquitin landscape

Protein ubiquitination is a powerful modulator of cellular functions. Classically linked to the degradation of proteins, it also plays a role in intracellular localization, DNA damage response, vesicle fusion events, and the immune and transcriptional responses. Ubiquitin is versatile and can code for several distinct signals, either by adding a single ubiquitin or forming a chain of ubiquitins on the target protein. The enzymatic cascade associated with the cellular process determines the nature of the modification. Numerous efforts have been made for the identification of ubiquitin acceptor sites in the target proteins using genetic, biochemical or mass-spectrometry based proteomic methods, such as affinity-based enrichment of ubiquitinated proteins, and antibody-based enrichment of modified peptides. Modern instrumentation enables quantitative mass-spectrometry strategies to identify and characterize hundreds of ubiquitin substrates in a single analysis making it the dominant method for ubiquitin site detection. Characterization of the inter-ubiquitin connectivity in ubiquitin polymers has also moved into focus, with the field of targeted proteomics techniques proving invaluable for identifying and quantifying linkage types found in such polyubiquitin chains. This review seeks to provide an overview of the many mass-spectrometry based proteomics techniques available for exploring this dynamic field

Tibial or hip BMD predict clinical fracture risk equally well: results from a prospective study in 700 elderly Swiss women

Summary: In a randomly selected cohort of Swiss community-dwelling elderly women prospectively followed up for 2.8 ± 0.6years, clinical fractures were assessed twice yearly. Bone mineral density (BMD) measured at tibial diaphysis (T-DIA) and tibial epiphysis (T-EPI) using dual-energy X-ray absorptiometry (DXA) was shown to be a valid alternative to lumbar spine or hip BMD in predicting fractures. Introduction: A study was carried out to determine whether BMD measurement at the distal tibia sites of T-EPI and T-DIA is predictive of clinical fracture risk. Methods: In a predefined representative cohort of Swiss community-dwelling elderly women aged 70-80years included in the prospective, multi-centre Swiss Evaluation of the Methods of Measurement of Osteoporotic Fracture risk (SEMOF) study, fracture risk profile was assessed and BMD measured at the lumbar spine (LS), hip (HIP) and tibia (T-DIA and T-EPI) using DXA. Thereafter, clinical fractures were reported in a bi-yearly questionnaire. Results: During 1,786 women-years of follow-up, 68 clinical fragility fractures occurred in 61 women. Older age and previous fracture were identified as risk factors for the present fractures. A decrease of 1 standard deviation in BMD values yielded a 1.5-fold (HIP) to 1.8-fold (T-EPI) significant increase in clinical fragility fracture hazard ratio (adjusted for age and previous fracture). All measured sites had comparable performance for fracture prediction (area under the curve range from 0.63 [LS] to 0.68 [T-EPI]). Conclusion: Fracture risk prediction with BMD measurements at T-DIA and T-EPI is a valid alternative to BMD measurements at LS or HIP for patients in whom these sites cannot be accessed for clinical, technical or practical reason

MicroRNA-34a promotes genomic instability by a broad suppression of genome maintenance mechanisms downstream of the oncogene KSHV-vGPCR

The Kaposi's sarcoma-associated herpesvirus (KSHV)-encoded chemokine receptor vGPCR acts as an oncogene in Kaposi's sarcomagenesis. Until now, the molecular mechanisms by which the vGPCR contributes to tumor development remain incompletely understood. Here, we show that the KSHV-vGPCR contributes to tumor progression through microRNA (miR)-34a-mediated induction of genomic instability. Large-scale analyses on the DNA, gene and protein level of cell lines derived from a mouse model of vGPCR-driven tumorigenesis revealed that a vGPCR-induced upregulation of miR-34a resulted in a broad suppression of genome maintenance genes. A knockdown of either the vGPCR or miR-34a largely restored the expression of these genes and confirmed miR-34a as a downstream effector of the KSHV-vGPCR that compromises genome maintenance mechanisms. This novel, protumorigenic role of miR-34a questions the use of miR-34a mimetics in cancer therapy as they could impair genome stability

The Future of Parenting Programs: II Implementation

This article examines the role that implementation science can play in evidence-based parenting programs. Although parenting programs can support parents in their caregiving roles, adapting and taking an evidence-based approach from one place to another without attending to implementation factors may contribute to poor impact in a new setting. Implementation science enables researchers to move beyond monitoring and evaluation of outcomes of a parenting program to understanding the process of putting the program into practice. Factors such as whether the program meets the needs of families and communities, how to secure buy-in from key stakeholders, what training and supervision are needed for the workforce, and ways that parenting programs can be integrated in existing infrastructure are all critical to successful implementation. Quality improvement can be built into the implementation process through feedback loops that inform rapid changes and testing cycles over time as a program is implemented. If researchers lead initial implementation of parenting programs, they must determine how the program can continue to work when using community workers and local systems rather than researchers. Open access components are especially important for the implementation of parenting programs in low- and middle-income countries to avoid prohibitive costs of proprietary programs and to benefit from flexibility in adapting components to meet the needs of particular local populations. Parenting programs benefit when policy makers, program leaders, and researchers attend not only to the what but also to the how of implementation.Objective.Design.Results.Conclusions

A generic C1C^1 map has no absolutely continuous invariant probability measure

Let $M$ be a smooth compact manifold (maybe with boundary, maybe disconnected) of any dimension $d \ge 1$. We consider the set of $C^1$ maps $f:M\to M$ which have no absolutely continuous (with respect to Lebesgue) invariant probability measure. We show that this is a residual (dense $G_\delta) set in the$C^1$ topology. In the course of the proof, we need a generalization of the usual Rokhlin tower lemma to non-invariant measures. That result may be of independent interest.Comment: 12 page

Sequential poly-ubiquitylation by specialized conjugating enzymes expands the versatility of a quality control ubiquitin ligase

The Doa10 quality control ubiquitin (Ub) ligase labels proteins with uniform lysine 48-linked poly-Ub (K48-pUB) chains for proteasomal degradation. Processing of Doa10 substrates requires the activity of two Ub conjugating enzymes. Here we show that the non-canonical conjugating enzyme Ubc6 attaches single Ub molecules not only to lysines but also to hydroxylated amino acids. These Ub moieties serve as primers for subsequent poly-ubiquitylation by Ubc7. We propose that the evolutionary conserved propensity of Ubc6 to mount Ub on diverse amino acids augments the number of ubiquitylation sites within a substrate and thereby increases the target range of Doa10. Our work provides new insights on how the consecutive activity of two specialized conjugating enzymes facilitates the attachment of poly-Ub to very heterogeneous client molecules. Such stepwise ubiquitylation reactions most likely represent a more general cellular phenomenon that extends the versatility yet sustains the specificity of the Ub conjugation system

Preoperative Cognitive Impairment and Postoperative Delirium Predict Decline in Activities of Daily Living after Cardiac Surgery-A Prospective, Observational Cohort Study.

Cardiac surgery and subsequent treatment in the intensive care unit (ICU) has been shown to be associated with functional decline, especially in elderly patients. Due to the different assessment tools and assessment periods, it remains yet unclear what parameters determine unfavorable outcomes. This study sought to identify risk factors during the entire perioperative period and focused on the decline in activity of daily living (ADL) half a year after cardiac surgery. Follow-ups of 125 patients were available. It was found that in the majority of patients (60%), the mean ADL declined by 4.9 points (95% CI, -6.4 to -3.5; p < 0.000). In the "No decline" -group, the ADL rose by 3.3 points (2.0 to 4.6; p < 0.001). A multiple regression analysis revealed that preoperative cognitive impairment (MMSE ≤ 26; Exp(B) 2.862 (95%CI, 1.192-6.872); p = 0.019) and duration of postoperative delirium ≥ 2 days (Exp(B) 3.534 (1.094-11.411); p = 0.035) was independently associated with ADL decline half a year after the operation and ICU. Of note, preoperative ADL per se was neither associated with baseline cognitive function nor a risk factor for functional decline. We conclude that the preoperative assessment of cognitive function, rather than functional assessments, should be part of risk stratification when planning complex cardiosurgical procedures

Quantitative proteomic analysis of Parkin substrates in Drosophila neurons.

Parkin (PARK2) is an E3 ubiquitin ligase that is commonly mutated in Familial Parkinson's Disease (PD). In cell culture models, Parkin is recruited to acutely depolarised mitochondria by PINK1. PINK1 activates Parkin activity leading to ubiquitination of multiple proteins, which in turn promotes clearance of mitochondria by mitophagy. Many substrates have been identified using cell culture models in combination with depolarising drugs or proteasome inhibitors, but not in more physiological settings.Here we utilized the recently introduced BioUb strategy to isolate ubiquitinated proteins in flies. Following Parkin Wild-Type (WT) and Parkin Ligase dead (LD) expression we analysed by mass spectrometry and stringent bioinformatics analysis those proteins differentially ubiquitinated to provide the first survey of steady state Parkin substrates using an in vivo model. We further used an in vivo ubiquitination assay to validate one of those substrates in SH-SY5Y cells.We identified 35 proteins that are more prominently ubiquitinated following Parkin over-expression. These include several mitochondrial proteins and a number of endosomal trafficking regulators such as v-ATPase sub-units, Syx5/STX5, ALiX/PDCD6IP and Vps4. We also identified the retromer component, Vps35, another PD-associated gene that has recently been shown to interact genetically with parkin. Importantly, we validated Parkin-dependent ubiquitination of VPS35 in human neuroblastoma cells.Collectively our results provide new leads to the possible physiological functions of Parkin activity that are not overtly biased by acute mitochondrial depolarisation

Regelung eines elastischen Fahrwegs unter Verwendung eines variablen Beobachters

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