The folding and aggregation properties of a single KH-domain protein: Ribosome binding factor A (RbfA) from Pseudomonas aeruginosa.

BACKGROUND: Ribosome-binding factor A from the pathogenic bacterium Pseudomonas aeruginosa (PaRbfA) is a small ribosome assembly factor, composed by a single KH domain, involved in the maturation of the 30S subunit. These domains are characterized by the ability to bind RNA or ssDNA and are often located in proteins involved in a variety of cellular functions. However, although the ability of proteins to fold properly, to misfold or to aggregate is of paramount importance for their cellular functions, limited information is available on these dynamic properties in the case of KH domains. METHODS: PaRbfA thermodynamic stability and folding mechanism: Far-UV CD and fluorescence spectroscopy, stopped-flow kinetics and chevron plot analysis, site-directed mutagenesis. Fibrils characterization: FT-IR spectroscopy, Thioflavin T fluorescence, Transmission Electron Microscopy (TEM) and X-ray fibrils diffraction. RESULTS: Quantitative analysis of the (un)folding kinetics of PaRbfA show that, in vitro, the protein folds via a 3-states mechanism involving a transiently populated folding intermediate. We also provide experimental evidences that PaRbfA can form ordered fibrils endowed with cross-ß structure even in mild conditions. CONCLUSION: These results lead to the hypothesis that the folding intermediate of PaRbfA may expose (some of) the predicted amyloidogenic regions, which could act as aggregation nuclei in the fibrillogenesis. GENERAL SIGNIFICANCE: The methodological approach presented herein could be readily adapted to verify the ability of other KH domain proteins to form cross-ß structured fibrils and to transiently populate a folding intermediate.

[Gynecological Lemierre's syndrome: A case report and literature review]. / Syndrome de Lemierre inversé : à propos d'un cas et revue de la littérature.

INTRODUCTION: Lemierre's syndrome is defined as an oropharyngeal infection due to Fusobacterium necrophorum, associated with septic thrombophlebitis of the internal jugular vein. The uncommon pelvic variant of the syndrome is a rare condition, poorly described in literature. CASE REPORT: We report a case of gynecological Lemierre's syndrome in a 19-year-old woman after a first sexual intercourse, who presented acute respiratory failure, left internal iliac vein thrombosis with pulmonary embolism, in the setting of salpingitis and F. necrophorum bacteriemia. CONCLUSION: Gynecological Lemierre's syndrome is a rare and unrecognized condition, which could be lethal. Early recognition of the disorder enables initiation of appropriate antibiotic therapy for 4 to 6 weeks, and discussion of anticoagulant therapy which indications are not yet well defined.

N6L pseudopeptide interferes with nucleophosmin protein-protein interactions and sensitizes leukemic cells to chemotherapy.

NPM1 is a multifunctional nucleolar protein implicated in several processes such as ribosome maturation and export, DNA damage response and apoptotic response to stress stimuli. The NPM1 gene is involved in human tumorigenesis and is found mutated in one third of acute myeloid leukemia patients, leading to the aberrant cytoplasmic localization of NPM1. Recent studies indicated that the N6L multivalent pseudopeptide, a synthetic ligand of cell-surface nucleolin, is also able to bind NPM1 with high affinity. N6L inhibits cell growth with different mechanisms and represents a good candidate as a novel anticancer drug for a number of malignancies of different histological origin. In this study we investigated whether N6L treatment could drive antitumor effect in acute myeloid leukemia cell lines. We found that N6L binds NPM1 at the N-terminal domain, co-localizes with cytoplasmic, mutated NPM1, and interferes with its protein-protein associations. N6L toxicity appears to be p53 dependent but interestingly, the leukemic cell line harbouring the mutated form of NPM1 is more resistant to treatment, suggesting that NPM1 cytoplasmic delocalization confers protection from p53 activation. Moreover, we show that N6L sensitizes AML cells to doxorubicin and cytarabine treatment. These studies suggest that N6L may be a promising option in combination therapies for acute myeloid leukemia treatment.

Structural investigation of nucleophosmin interaction with the tumor suppressor Fbw7γ.

Nucleophosmin (NPM1) is a multifunctional nucleolar protein implicated in ribogenesis, centrosome duplication, cell cycle control, regulation of DNA repair and apoptotic response to stress stimuli. The majority of these functions are played through the interactions with a variety of protein partners. NPM1 is frequently overexpressed in solid tumors of different histological origin. Furthermore NPM1 is the most frequently mutated protein in acute myeloid leukemia (AML) patients. Mutations map to the C-terminal domain and lead to the aberrant and stable localization of the protein in the cytoplasm of leukemic blasts. Among NPM1 protein partners, a pivotal role is played by the tumor suppressor Fbw7γ, an E3-ubiquitin ligase that degrades oncoproteins like c-MYC, cyclin E, Notch and c-jun. In AML with NPM1 mutations, Fbw7γ is degraded following its abnormal cytosolic delocalization by mutated NPM1. This mechanism also applies to other tumor suppressors and it has been suggested that it may play a key role in leukemogenesis. Here we analyse the interaction between NPM1 and Fbw7γ, by identifying the protein surfaces implicated in recognition and key aminoacids involved. Based on the results of computational methods, we propose a structural model for the interaction, which is substantiated by experimental findings on several site-directed mutants. We also extend the analysis to two other NPM1 partners (HIV Tat and CENP-W) and conclude that NPM1 uses the same molecular surface as a platform for recognizing different protein partners. We suggest that this region of NPM1 may be targeted for cancer treatment.

ATP2C1 gene mutations in Hailey-Hailey disease and possible roles of SPCA1 isoforms in membrane trafficking.

ATP2C1 gene codes for the secretory pathway Ca(2+)/Mn(2+)-ATPase pump type 1 (SPCA1) localizing at the golgi apparatus. Mutations on the human ATP2C1 gene, causing decreased levels of the SPCA1 expression, have been identified as the cause of the Hailey-Hailey disease, a rare skin disorder. In the last few years, several mutations have been described, and here we summarize how they are distributed along the gene and how missense mutations affect protein expression. SPCA1 is expressed in four different isoforms through alternative splicing of the ATP2C1 gene and none of these isoforms is differentially affected by any of these mutations. However, a better understanding of the tissue specific expression of the isoforms, their localization along the secretory pathway, their specific binding partners and the role of the C-terminal tail making isoforms different from each other, will be future goals of the research in this field.

Functional and structural analysis of four novel mutations of CYP21A2 gene in Italian patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.

The fine-tuning of TRAF2-GSTP1-1 interaction: effect of ligand binding and in situ detection of the complex.

We provide the first biochemical evidence of a direct interaction between the glutathione transferase P1-1 (GSTP1-1) and the TRAF domain of TNF receptor-associated factor 2 (TRAF2), and describe how ligand binding modulates such an equilibrium. The dissociation constant of the heterocomplex is K(d)=0.3 µM; however the binding affinity strongly decreases when the active site of GSTP1-1 is occupied by the substrate GSH (K(d)≥2.6 µM) or is inactivated by oxidation (Kd=1.7 µM). This indicates that GSTP1-1's TRAF2-binding region involves the GSH-binding site. The GSTP1-1 inhibitor NBDHEX further decreases the complex's binding affinity, as compared with when GSH is the only ligand; this suggests that the hydrophobic portion of the GSTP1-1 active site also contributes to the interaction. We therefore hypothesize that TRAF2 binding inactivates GSTP1-1; however, analysis of the data, using a model taking into account the dimeric nature of GSTP1-1, suggests that GSTP1-1 engages only one subunit in the complex, whereas the second subunit maintains the catalytic activity or binds to other proteins. We also analyzed GSTP1-1's association with TRAF2 at the cellular level. The TRAF2-GSTP1-1 complex was constitutively present in U-2OS cells, but strongly decreased in S, G2 and M phases. Thus the interaction appears regulated in a cell cycle-dependent manner. The variations in the levels of individual proteins seem too limited to explain the complex's drastic decline observed in cells progressing from the G0/G1 to the S-G2-M phases. Moreover, GSH's intracellular content was so high that it always saturated GSTP1-1. Interestingly, the addition of NBDHEX maintains the TRAF2-GSTP1-1 complex at low levels, thus causing a prolonged cell cycle arrest in the G2/M phase. Overall, these findings suggest that a reversible sequestration of TRAF2 into the complex may be crucial for cell cycle progression and that multiple factors are involved in the fine-tuning of this interaction.

[A not so common "community" acquired pneumonia]. / Une pneumopathie "communautaire" pas si commune que ça!

Pulmonary actinomycosis is a rare infection, the clinical presentation of which usually suggests tuberculosis or pulmonary neoplasia. We report the case of a 50-year-old male patient, with a peculiar initial clinical presentation mimicking acute community acquired pneumonia but with an atypical evolution leading to a final diagnosis of actinomycosis.

Value of (18)F-FDG-PET/CT in patients with fever of unknown origin and unexplained prolonged inflammatory syndrome: a single centre analysis experience.

OBJECTIVE: The aim of our study was to evaluate the diagnostic contribution of (18)F-fluoro-deoxyglucose ((18)F-FDG)-positron emission tomography (PET)/computed tomography (CT) in patients with fever of unknown origin (FUO) or unexplained prolonged inflammatory syndrome (UPIS) in real life. PATIENTS AND METHODS: We performed a retrospective study including 14 patients with FUO or UPIS hospitalised in our institution (Strasbourg University Hospital, France) between January 2005 and July 2006. (18)F-FDG-PET/CT was considered helpful when abnormal results allowed an accurate diagnosis. RESULTS: (18)F-FDG-PET/CT was helpful in half the patients (7/14) for final diagnosis. A diagnosis was reached in 87.5% of the patients (7/8) with an abnormal (18)F-FDG-PET/CT but only in 50% of the patients (3/6) with a normal (18)F-FDG-PET/CT. Conventional chest and abdominal CT was performed in 13 patients before ordering (18)F-FDG-PET/CT. We considered that (18)F-FDG-PET/CT was essential to establish the final diagnosis in only 23% of the patients (3/13) since neither chest nor abdominal CT identified abnormalities consistent with the final diagnosis. However, among the three patients, two were diagnosed with large vessel vasculitis and one patient with local prosthetic infection. CONCLUSIONS: Our study supports the potential interest of (18)F-FDG-PET/CT in the diagnostic workup of FUO and UPIS as it helped establish a fine diagnosis in half of the cases. However, (18)F-FDG-PET/CT appeared to be essential to the final diagnosis in only 23% of the cases. In our opinion, this protocol should be performed as a second level test, especially when conventional CT is normal or is unable to discriminate between active and silent lesions.

[Cytomegalovirus infection in an immunocompetent patient complicated with pulmonary embolism and mimicking giant cell arteritis associated with polymyalgia rheumatica]. / Embolie pulmonaire compliquant une infection à cytomégalovirus, infection mimant une maladie de Horton associée à une pseudopolyarthrite rhizomélique chez un sujet immunocompétent.

Cytomegalovirus infection is generally asymptomatic in immunocompetent patients but can also provide a wide spectrum of clinical and biological signs. We report the case of a cytomegalovirus infection in an immunocompetent adult mimicking giant cell arteritis associated with polymyalgia rheumatica and complicated with pulmonary embolism. We review and discuss the venous prothrombic properties of CMV in immunocompetent patients.

[Drug-induced agranulocytosis in rheumatology. A retrospective study of 12 cases]. / Agranulocytoses médicamenteuses en rhumatologie : étude rétrospective de 12 cas.

OBJECTIVE: the aim of this study is to determine the clinical characteristics of drug-induced agranulocytosis in rheumatology. PATIENTS AND METHOD: it is a retrospective monocentric study, including all cases of rheumatologic drug-induced agranulocytosis followed between January 1985 and December 2005. RESULTS: eight female and 4 men, mean age 62.5 years (range: 17-79), were included in the present study. The causative drugs were: anti-inflammatory agents (n=5, 41.6%), methotrexate (n=3, 25%), sulfasalazine (n=2, 16.6%), noramidopyrine (n=1) and dapsone (n=1). Main clinical features included infectious disorders in 9 cases (75%) with 4 cases of septicemia (33.3%) and 2 cases of septic shock (16.6%). The mean neutrophils count was 0.132 x 10(9)/L (range, 0-0.4). Outcome was favorable in 11 patients (91.6%). One patient died of septic complications. CONCLUSION: in rheumatology, drug-induced agranulocytosis is a rare but life-threatening disorder.

[Pancytopenia related to low-dose methotrexate: study of five cases and review of the literature]. / Pancytopénie sous méthotrexate à faibles doses: étude de cinq observations et revue de la littérature.

PURPOSE: The aim of this study is to report personal experience of pancytopenia related to low-dose methotrexate and to review the literature. METHODS: We included retrospectively all cases of pancytopenia related to low-dose methotrexate (<25 mg/week), followed between January 1997 and December 2006, in the university hospital of Strasbourg, France. RESULTS: Five women, mean age 75.6 years, were included in the present study. Clinical manifestations included: symptomatic anemia (n=4), infection (n=3) and hemorrhagic manifestations (n=2); one patient had no feature. Mean hemoglobin concentration was 8,8 g/dl; mean white cell and platelet counts were 1,500 /mm(3) and 16,000 /mm(3), respectively. Potential risk factors were identified in all patients: renal failure and low serum albumin levels (n=5), anti-inflammatory drug intake (n=2), folate deficiency (n=4) and cobalamin deficiency (n=1). One patient died of septic and hemorrhagic cerebral complications. CONCLUSION: Pancytopenia related to tow-dose methotrexate is a rare but life-threatening disorder. Search and prevention of potential risk factors are required in all patients; determination of MTHFR genotype may be of several interests as folate supplementation.

[Update of clinical findings in cobalamin deficiency: personal data and review of the literature]. / Manifestations hématologiques de la carence en vitamine B12: données personnelles et revue de la littérature.

PURPOSE: During last decades, several progresses have been made in the diagnosis of cobalamin (vitamin B12) deficiency. Routine used of cobalamin standardized assays have potentially modified the frequency and the type of hematologic abnormalities. CURRENT KNOWLEDGE AND KEY POINTS: Current studies on cobalamin deficiency, including more precise definitions and the description of new etiologies of cobalamin deficiency in adults, as the food-cobalamin malabsorption syndrome, show that hematological abnormalities are generally incomplete compared to historical descriptions of megaloblastic anemia. Nevertheless, they include severe manifestations in 10% of the patients: pancytopenia, severe anemia (hemoglobin < 6 g/dl) or hemolytic anemia and pseudo thrombotic microangiopathy related to cobalamin deficiency. These studies also show the efficacy of new treatment modalities including oral cobalamin administration. PROSPECTS AND PROJECTS: Future studies will confirm these data with the routine use of the new cobalamin assay: holotranscobalamin and validate the usefulness of oral cobalamin therapy.

[Bilateral corneal endothelial decompensation after postsepticemia coma]. / Décompensation endothéliale bilatérale après coma post septicémie à staphylocoque.

We report a case of unexplained bilateral corneal endothelial decompensation after a coma. A 71-year-old man with no medical history presented with bilateral endothelial decompensation that required penetrating keratoplasty of the left eye combined with cataract surgery. This coma was caused by septicemia originally due to staphylococcus infection following catheter placement in preparation for a CT scan. Visual acuity of the left eye was 20/400 and 20/100 in the right eye when the patient awoke from the coma. After examination, we noted only stromal thickening and Descemet membrane folds causing corneal edema predominating OS. The rest of the exam was normal. Six months after surgery, visual acuity improved to 20/25. The most probable physiopathological mechanism of this decompensation is an iatrogenic complication from drugs administered during the patient's stay in intensive care (oxacillin), but we cannot rule out direct aggression of a bacterium or its toxin or the decompensation of a preexisting pathology.

A decision tree for genetic diagnosis of hereditary periodic fever in unselected patients.

BACKGROUND: The diagnostic value of molecular analysis of the familial Mediterranean fever (FMF) gene (Mediterranean fever (MEFV)) has been well established only in patients selected on the basis of ethnic background or clinical criteria. Genetic diagnosis for other hereditary periodic fever syndromes has been poorly evaluated. OBJECTIVE: To determine the diagnostic contribution of genetic tests for hereditary periodic syndromes in a large, unselected series of patients. METHODS: A retrospective study was conducted on 1941 patients referred to us for FMF genetic tests between 1997 and 2005. MEFV genotypes were compared with clinical data to appraise criteria for FMF diagnosis. Genetic tests for tumour necrosis factor receptor-associated periodic syndrome (TRAPS), hyperimmunoglobulinaemia D syndrome (HIDS) and cryopyrin-associated periodic syndromes (CAPS) were also reviewed. RESULTS: 71% of the 1574 patients with enough data had a clinical diagnosis of FMF according to the widely used Israeli criteria. Two MEFV mutations were found in only 409 patients of this subgroup (sensitivity = 37%) and in 15 (3.3%) of the patients with an improbable clinical diagnosis of FMF (specificity = 97%). Molecular diagnosis for alternate hereditary periodic syndromes was carried out in 456 of the patients having a non-conclusive FMF genetic test. A positive diagnosis was obtained in 31 of these patients (TRAPS (n = 19), HIDS (n = 4) and CAPS (n = 8)). CONCLUSIONS: First-line MEFV mutation screening in patients with clinically typical FMF may be appropriate only in particular areas. To optimise genetic diagnosis, we propose a decision tree, which, with the advice of an expert practitioner, could help redirect test indications towards non-FMF hereditary periodic syndromes.

The crystal structure of polygalacturonase-inhibiting protein (PGIP), a leucine-rich repeat protein involved in plant defense.

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that protect plants from fungal invasion. They interact with endopolygalacturonases secreted by phytopathogenic fungi, inhibit their enzymatic activity, and favor the accumulation of oligogalacturonides, which activate plant defense responses. PGIPs are members of the leucine-rich repeat (LRR) protein family that in plants play crucial roles in development, defense against pathogens, and recognition of beneficial microbes. Here we report the crystal structure at 1.7-A resolution of a PGIP from Phaseolus vulgaris. The structure is characterized by the presence of two beta-sheets instead of the single one originally predicted by modeling studies. The structure also reveals a negatively charged surface on the LRR concave face, likely involved in binding polygalacturonases. The structural information on PGIP provides a basis for designing more efficient inhibitors for plant protection.