Publications (7)22.94 Total impact
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Article: Strategies to identify recognition signals and targets of SUMOylation Elisa Da Silva-Ferrada, Fernando Lopitz-Otsoa, Valerie Lang, Manuel S. Rodríguez, and Rune Matthiesen Received 9 March 2012; Accepted 12 April 2012Academic E
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ABSTRACT: Received 9 March 2012; Accepted 12 April 2012Academic Editor: Philip CoffinoBiochemistry Research International. 01/2012; -
Article: Strategies to identify recognition signals and targets of SUMOylation
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ABSTRACT: SUMOylation contributes to the regulation of many essential cellular factors. Diverse techniques have been used to explore the functional consequences of protein SUMOylation. Most approaches consider the identification of sequences on substrates, adaptors or receptors regulating the SUMO conjugation, recognition or de-conjugation. The large majority of the studied SUMOylated proteins contain the sequence [IVL]KxE. SUMOylated proteins are recognized by at least 3 types of hydrophobic SUMO-interacting motifs (SIMs) that contribute to coordinate SUMO-dependent functions. Typically, SIMs are constituted by a hydrophobic core flanked by one or two clusters of negatively charged amino acid residues. Multiple SIMs can integrate SUMO binding domains (SBD), optimizing binding and control over SUMO-dependent processes. Here we present a survey of the methodologies used to study SUMO-regulated functions helping to the identification of cis and trans sequences controlling SUMOylation. Furthermore an integrative analysis of known and putative SUMO substrates illustrates an updated landscape of several SUMO-regulated events. The strategies and analysis presented here should contribute to the understanding of SUMO-controlled functions and provide rational approach to identify biomarkers or choose possible targets for intervention in processes were SUMOylation plays a critical role.Biochemistry Research International. 01/2012; -
Article: Oligomerization conditions Mdm2-mediated efficient p53 polyubiquitylation but not its proteasomal degradation.
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ABSTRACT: In normal cells p53 is maintained at low level through the action of the ubiquitin-proteasome system. As a consequence of p53 transcriptional activity, various regulators of this tumor suppressor are produced, forming a negative feedback loop tightly controlling p53 stability. One of the most prominent is the ubiquitin-ligase Mdm2. Here, we have used a transfer of signals strategy to study the p53 degradation process promoted by Mdm2 in the absence of p53 transcriptional activity. Our results show that in a p53 null background, transcriptionally silent p53-fusions require multiple N- and C-terminal signals to be optimally targeted to proteasomal degradation. As for WT p53, p53-fusions able to form tetramers are polyubiquitylated and optimally degraded by the proteasome. However, p53 molecules unable to oligomerize, show Mdm2-mediated polyubiquitylation deficiency but are still targeted to proteasome degradation in vitro and ex vivo. In the presence of Mdm2, nuclear shuttling of p53 monomeric fusions favours proteasome-dependent degradability but not its polyubiquitylation. In vitro, 26S proteasome fails to drive degradation of OD mutants in the presence of Mdm2, suggesting the contribution of additional cellular factors in this process. All together, our results indicate that Mdm2-mediated proteasome-dependent degradation of polyubiquitylation deficient p53 monomers is mechanistically possible, taking alternative pathways to better achieve their proteolysis. These results support the existence of additional levels to regulate p53 stability and activity acting on individual subunits of the functional tetramer.The international journal of biochemistry & cell biology 05/2010; 42(5):725-35. · 4.89 Impact Factor -
Article: Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities.
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ABSTRACT: Post-translational modification with ubiquitin is one of the most important mechanisms in the regulation of protein stability and function. However, the high reversibility of this modification is the main obstacle for the isolation and characterization of ubiquitylated proteins. To overcome this problem, we have developed tandem-repeated ubiquitin-binding entities (TUBEs) based on ubiquitin-associated (UBA) domains. TUBEs recognize tetra-ubiquitin with a markedly higher affinity than single UBA domains, allowing poly-ubiquitylated proteins to be efficiently purified from cell extracts in native conditions. More significant is the fact that TUBEs protect poly-ubiquitin-conjugated proteins, such as p53 and IkappaBalpha, both from proteasomal degradation and de-ubiquitylating activity present in cell extracts, as well as from existing proteasome and cysteine protease inhibitors. Therefore, these new 'molecular traps' should become valuable tools for purifying endogenous poly-ubiquitylated proteins, thus contributing to a better characterization of many essential functions regulated by these post-translational modifications.EMBO Reports 10/2009; 10(11):1250-8. · 7.36 Impact Factor -
Article: Association between hand-off patients and subject exam performance in medicine clerkship students.
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ABSTRACT: Teaching hospitals increasingly rely on transfers of patient care to another physician (hand-offs) to comply with duty hour restrictions. Little is known about the impact of hand-offs on medical students. To evaluate the impact of hand-offs on the types of patients students see and the association with their subsequent Medicine Subject Exam performance. Observational study over 1 year. Third-year medical students in an Inpatient Medicine Clerkship at five hospitals with night float systems. Primary outcome: Medicine Subject Exam at the end of the clerkship; explanatory variables: number of fresh (without prior evaluation) and hand-off patients, diagnoses, subspecialty patients, and full evaluations performed during the clerkship, and United Stated Medical Licensing Examination (USMLE) Step I scores. Of the 2,288 patients followed by 89 students, 990 (43.3%) were hand-offs. In a linear regression model, the only variables significantly associated with students' Subject Exam percentile rankings were USMLE Step I scores (B = 0.26, P < 0.001) and the number of full evaluations completed on fresh patients (B =0.20, P = 0.048; model r (2) = 0.58). In other words, for each additional fresh patient evaluated, Subject Exam percentile rankings increased 0.2 points. For students in the highest quartile of Subject Exam percentile rankings, only Step I scores showed a significant association (B = 0.22, P = 0.002; r (2) = 0.5). For students in the lowest quartile, only fresh patient evaluations demonstrated a significant association (B = 0.27, P = 0.03; r (2) = 0.34). Hand-offs constitute a substantial portion of students' patients and may have less educational value than "fresh" patients, especially for lower performing students.Journal of General Internal Medicine 08/2009; 24(9):1018-22. · 2.83 Impact Factor -
Article: The effect of nonteaching services on the distribution of inpatient cases for internal medicine residents.
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ABSTRACT: Nonteaching services (NTSs) are becoming increasingly prevalent in academic hospitals. This study was designed to determine whether the presence of an NTS is associated with higher acuity and altered case mix on the teaching service. The authors carried out a retrospective, cross-sectional analysis of data about all general medical admissions between January 1, 2005 and June 30, 2005 to either of two teaching hospitals in Rochester, New York. A total of 6,907 inpatients were studied, of whom 1,976 (29%) were admitted to medicine resident services and 4,931 (71%) were admitted to NTSs. Hospital billing databases were used to determine patient demographics, ICD-9 diagnoses, Charlson Comorbidity Index scores, and patient disposition. Compared with NTS patients, patients on resident services had higher median Charlson Comorbidity Index scores (3.0 versus 2.0, P < .001) and numbers of comorbidities (9.0 versus 8.0, P < .001) and were more likely to require intensive care (15.5% versus 7.6%, P < .001) and to die in the hospital (8.2% versus 4.5%, P < .001). Patients on the resident services were more likely to have acute renal failure, respiratory failure, septicemia, and HIV. Residents were less likely to care for patients with primary diagnoses of chest pain, cellulitis, alcohol withdrawal, and sickle cell crisis. The differences in patients' conditions between resident services and NTSs were similar in the two hospitals and also among patients who had not received intensive care. Patients on resident services may be more medically complex and more likely to have high-acuity diagnoses than patients on NTSs. How these differences affect residents' education, residents' career decisions, and practice styles deserves further study.Academic medicine: journal of the Association of American Medical Colleges 03/2009; 84(2):220-5. · 2.34 Impact Factor -
Article: betaTrCP-mediated proteolysis of NF-kappaB1 p105 requires phosphorylation of p105 serines 927 and 932.
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ABSTRACT: NF-kappaB1 p105 functions both as a precursor of NF-kappaB1 p50 and as a cytoplasmic inhibitor of NF-kappaB. Following the stimulation of cells with tumor necrosis factor alpha (TNF-alpha), the IkappaB kinase (IKK) complex rapidly phosphorylates NF-kappaB1 p105 on serine 927 in the PEST region. This phosphorylation is essential for TNF-alpha to trigger p105 degradation, which releases the associated Rel/NF-kappaB subunits to translocate into the nucleus and regulate target gene transcription. Serine 927 resides in a conserved motif (Asp-Ser(927)-Gly-Val-Glu-Thr-Ser(932)) homologous to the IKK target sequence in IkappaBalpha. In this study, TNF-alpha-induced p105 proteolysis was revealed to additionally require the phosphorylation of serine 932. Experiments with IKK1(-/-) and IKK2(-/-) double knockout embryonic fibroblasts demonstrate that the IKK complex is essential for TNF-alpha to stimulate phosphorylation on p105 serines 927 and 932. Furthermore, purified IKK1 and IKK2 can each phosphorylate a glutathione S-transferase-p105(758-967) fusion protein on both regulatory serines in vitro. IKK-mediated p105 phosphorylation generates a binding site for betaTrCP, the receptor subunit of an SCF-type ubiquitin E3 ligase, and depletion of betaTrCP by RNA interference blocks TNF-alpha-induced p105 ubiquitination and proteolysis. Phosphopeptide competition experiments indicate that betaTrCP binds p105 more effectively when both serines 927 and 932 are phosphorylated. Interestingly, however, betaTrCP affinity for the IKK-phosphorylated sequence on p105 is substantially lower than that on IkappaBalpha. Thus, it appears that reduced p105 recruitment of betaTrCP and subsequent ubiquitination may contribute to delayed p105 proteolysis after TNF-alpha stimulation relative to that for IkappaBalpha.Molecular and Cellular Biology 02/2003; 23(1):402-13. · 5.53 Impact Factor
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2003
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MRC National Institute for Medical Research
- Division of Immune Cell Biology
London, ENG, United Kingdom
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