Old and new inhibitors of quinone reductase 2

Quinone reductase 2 is a cytosolic enzyme which catalyses the reduction of quinones, such as menadione and coenzymes Q. Despite a relatively close sequence-based resemblance to NAD(P)H:quinone oxidoreductase 1 (QR1), it has many different features. QR2 is the third melatonin binding site (MT3). It is inhibited in the micromolar range by melatonin, and does not accept conventional phosphorylated nicotinamides as hydride donors. QR2 has a powerful capacity to activate quinones leading to unexpected toxicity situations. In the present paper, we report the characterization of three QR2 modulators: melatonin, resveratrol and S29434. The latter compound inhibits QR2 activity with an IC₅₀ in the low nanomolar range. The potency of the modulators ranged as follows, from the least to the most potent: melatonin < resveratrol < S29434. These molecular tools might permit to explore and better understand the relationship existing between QR2 catalytic activity and the various pathological situations in which QR2 has a key role.     

Phylogenetic and mutational analyses reveal key residues for UDP-glucuronic acid binding and activity of beta1,3-glucuronosyltransferase I (GlcAT-I)

The beta1,3-glucuronosyltransferases are responsible for the completion of the protein-glycosaminoglycan linkage region of proteoglycans and of the HNK1 epitope of glycoproteins and glycolipids by transferring glucuronic acid from UDP-alpha-D-glucuronic acid (UDP-GlcA) onto a terminal galactose residue. Here, we develop phylogenetic and mutational approaches to identify critical residues involved in UDP-GlcA binding and enzyme activity of the human beta1,3-glucuronosyltransferase I (GlcAT-I), which plays a key role in glycosaminoglycan biosynthesis. Phylogeny analysis identified 119 related beta1,3-glucuronosyltransferase sequences in vertebrates, invertebrates, and plants that contain eight conserved peptide motifs with 15 highly conserved amino acids. Sequence homology and structural information suggest that Y84, D113, R156, R161, and R310 residues belong to the UDP-GlcA binding site. The importance of these residues is assessed by site-directed mutagenesis, UDP affinity and kinetic analyses. Our data show that uridine binding is primarily governed by stacking interactions with the phenyl group of Y84 and also involves interactions with aspartate 113. Furthermore, we found that R156 is critical for enzyme activity but not for UDP binding, whereas R310 appears less important with regard to both activity and UDP interactions. These results clearly discriminate the function of these two active site residues that were predicted to interact with the pyrophosphate group of UDP-GlcA. Finally, mutation of R161 severely compromises GlcAT-I activity, emphasizing the major contribution of this invariant residue. Altogether, this phylogenetic approach sustained by biochemical analyses affords new insight into the organization of the beta1,3-glucuronosyltransferase family and distinguishes the respective importance of conserved residues in UDP-GlcA binding and activity of GlcAT-I.     

Huntingtin proteolysis releases non‐polyQ fragments that cause toxicity through dynamin 1 dysregulation

Cleavage of mutant huntingtin (HTT) is an essential process in Huntington's disease (HD), an inherited neurodegenerative disorder. Cleavage generates N‐ter fragments that contain the polyQ stretch and whose nuclear toxicity is well established. However, the functional defects induced by cleavage of full‐length HTT remain elusive. Moreover, the contribution of non‐polyQ C‐terminal fragments is unknown. Using time‐ and site‐specific control of full‐length HTT proteolysis, we show that specific cleavages are required to disrupt intramolecular interactions within HTT and to cause toxicity in cells and flies. Surprisingly, in addition to the canonical pathogenic N‐ter fragments, the C‐ter fragments generated, that do not contain the polyQ stretch, induced toxicity via dilation of the endoplasmic reticulum (ER) and increased ER stress. C‐ter HTT bound to dynamin 1 and subsequently impaired its activity at ER membranes. Our findings support a role for HTT on dynamin 1 function and ER homoeostasis. Proteolysis‐induced alteration of this function may be relevant to disease.     

Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.     

Xanthomonas T3S Effector XopN Suppresses PAMP-Triggered Immunity and Interacts with a Tomato Atypical Receptor-Like Kinase and TFT1

XopN is a virulence factor from Xanthomonas campestris pathovar vesicatoria (Xcv) that is translocated into tomato (Solanum lycopersicum) leaf cells by the pathogen''s type III secretion system. Xcv ΔxopN mutants are impaired in growth and have reduced ability to elicit disease symptoms in susceptible tomato leaves. We show that XopN action in planta reduced pathogen-associated molecular pattern (PAMP)-induced gene expression and callose deposition in host tissue, indicating that XopN suppresses PAMP-triggered immune responses during Xcv infection. XopN is predicted to have irregular, α-helical repeats, suggesting multiple protein–protein interactions in planta. Consistent with this prediction, XopN interacted with the cytosolic domain of a Tomato Atypical Receptor-Like Kinase1 (TARK1) and four Tomato Fourteen-Three-Three isoforms (TFT1, TFT3, TFT5, and TFT6) in yeast. XopN/TARK1 and XopN/TFT1 interactions were confirmed in planta by bimolecular fluorescence complementation and pull-down analysis. Xcv ΔxopN virulence defects were partially suppressed in transgenic tomato leaves with reduced TARK1 mRNA levels, indicating that TARK1 plays an important role in the outcome of Xcv–tomato interactions. These data provide the basis for a model in which XopN binds to TARK1 to interfere with TARK1-dependent signaling events triggered in response to Xcv infection.     

Short- and long-term effects of anti-CD20 treatment on B cell ontogeny in bone marrow of patients with rheumatoid arthritis

Introduction  

In the present study we evaluated changes in the B cell phenotype in peripheral blood and bone marrow (BM) of patients with rheumatoid arthritis (RA) following anti-CD20 treatment using rituximab.     

Intracellular trafficking of TRP channels

Thirteen years ago, it was suggested that exocytotic insertion of store-operated channels into the plasma membrane lead to increased Ca(2+) entry in non-excitable cells upon G protein-coupled or tyrosine kinase receptor stimulation. Since the discovery of the TRP channel superfamily and their involvement in receptor-induced Ca(2+) entry, many studies have shown that different members of the TRP superfamily translocate into the plasma membrane upon stimulation. While the exact molecular mechanism by which TRP channels insert into the plasma membrane is unknown, TRP-binding proteins have been shown to directly regulate this trafficking. This review summarizes recent advances related to the mechanism of TRP channel trafficking, focusing on the role of TRP-binding proteins.     

Natural History of Cardiac and Respiratory Involvement,Prognosis and Predictive Factors for Long-Term Survival in Adult Patients with Limb Girdle Muscular Dystrophies Type 2C and 2D

BackgroundType 2C and 2D limb girdle muscular dystrophies (LGMD) are a group of autosomal recessive limb girdle muscular dystrophies manifested by proximal myopathy, impaired respiratory muscle function and cardiomyopathy. The correlation and the prognostic impact of respiratory and heart impairment are poorly described. We aimed to describe the long-term cardiac and respiratory follow-up of these patients and to determine predictive factors of cardio-respiratory events and mortality in LGMD 2C and 2D.MethodsWe reviewed the charts of 34 LGMD patients, followed from 2005 to 2015, to obtain echocardiographic, respiratory function and sleep recording data. We considered respiratory events (acute respiratory failure, pulmonary sepsis, atelectasis or pneumothorax), cardiac events (acute heart failure, significant cardiac arrhythmia or conduction block, ischemic stroke) and mortality as outcomes of interest for the present analysis.ResultsA total of 21 patients had type 2C LGMD and 13 patients had type 2D. Median age was 30 years [IQR 24–38]. At baseline, median pulmonary vital capacity (VC) was 31% of predicted value [20–40]. Median maximal inspiratory pressure (MIP) was 31 cmH₂O [IQR 20.25–39.75]. Median maximal expiratory pressure (MEP) was 30 cm H₂O [20–36]. Median left ventricular ejection fraction (LVEF) was 55% [45–64] with 38% of patients with LVEF <50%. Over a median follow-up of 6 years, we observed 38% respiratory events, 14% cardiac events and 20% mortality. Among baseline characteristics, LVEF and left ventricular end diastolic diameter (LVEDD) were associated with mortality, whilst respiratory parameters (VC, MIP, MEP) and the need for home mechanical ventilation (HMV) were associated with respiratory events.ConclusionIn our cohort of severely respiratory impaired type 2C and 2D LGMD, respiratory morbidity was high. Cardiac dysfunction was frequent in particular in LGMD 2C and had an impact on long-term mortality.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT02501083","term_id":"NCT02501083"}}NCT02501083     

Learning to Eat Vegetables in Early Life: The Role of Timing,Age and Individual Eating Traits

Vegetable intake is generally low among children, who appear to be especially fussy during the pre-school years. Repeated exposure is known to enhance intake of a novel vegetable in early life but individual differences in response to familiarisation have emerged from recent studies. In order to understand the factors which predict different responses to repeated exposure, data from the same experiment conducted in three groups of children from three countries (n = 332) aged 4–38 m (18.9±9.9 m) were combined and modelled. During the intervention period each child was given between 5 and 10 exposures to a novel vegetable (artichoke puree) in one of three versions (basic, sweet or added energy). Intake of basic artichoke puree was measured both before and after the exposure period. Overall, younger children consumed more artichoke than older children. Four distinct patterns of eating behaviour during the exposure period were defined. Most children were “learners” (40%) who increased intake over time. 21% consumed more than 75% of what was offered each time and were labelled “plate-clearers”. 16% were considered “non-eaters” eating less than 10 g by the 5^th exposure and the remainder were classified as “others” (23%) since their pattern was highly variable. Age was a significant predictor of eating pattern, with older pre-school children more likely to be non-eaters. Plate-clearers had higher enjoyment of food and lower satiety responsiveness than non-eaters who scored highest on food fussiness. Children in the added energy condition showed the smallest change in intake over time, compared to those in the basic or sweetened artichoke condition. Clearly whilst repeated exposure familiarises children with a novel food, alternative strategies that focus on encouraging initial tastes of the target food might be needed for the fussier and older pre-school children.