Physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathies

Some physico-chemical properties of R140G and K141Q mutants of human small heat shock protein HspB1 associated with hereditary peripheral neuropathy were analyzed. Mutation K141Q did not affect intrinsic Trp fluorescence and interaction with hydrophobic probe bis-ANS, whereas mutation R140G decreased both intrinsic fluorescence and fluorescence of bis-ANS bound to HspB1. Both mutations decreased thermal stability of HspB1. Mutation R140G increased, whereas mutation K141Q decreased the rate of trypsinolysis of the central part (residues 5–188) of HspB1. Both the wild type HspB1 and its K141Q mutant formed large oligomers with apparent molecular weight ∼560 kDa. The R140G mutant formed two types of oligomers, i.e. large oligomers tending to aggregate and small oligomers with apparent molecular weight ∼70 kDa. The wild type HspB1 formed mixed homooligomers with R140G mutant with apparent molecular weight ∼610 kDa. The R140G mutant was unable to form high molecular weight heterooligomers with HspB6, whereas the K141Q mutant formed two types of heterooligomers with HspB6. In vitro measured chaperone-like activity of the wild type HspB1 was comparable with that of K141Q mutant and was much higher than that of R140G mutant. Mutations of homologous hot-spot Arg (R140G of HspB1 and R120G of αB-crystallin) induced similar changes in the properties of two small heat shock proteins, whereas mutations of two neighboring residues (R140 and K141) induced different changes in the properties of HspB1.     

A 5q12.1–5q12.3 microdeletion in a case with a balanced exceptional complex chromosomal rearrangement

Complex chromosomal rearrangements are very rare chromosomal abnormalities. Individuals with a complex chromosomal rearrangement can be phenotypically normal or display a clinical abnormality. It is believed that these abnormalities are due to either microdeletions or microduplications at the translocation breakpoints or as a result of disruption of the genes located in the breakpoints. In this study we describe a 2-year-old child with mental retardation and developmental delay in whom a de novo apparently balanced exceptional complex chromosomal rearrangement was found through conventional cytogenetic analysis. Using both cytogenetic and FISH analysis, the patient's karyotype was found to be: 46,XY,der(5)t(5;7)(p15.1;7q34),t(5;8)(q13.1;8q24.1)dn. A large, clinically significant deletion which encompassed 887.69 kb was detected at the 5q12.1–5q12.3 (chr5:62.886.523–63.774.210) genomic region using array-CGH. This deleted region includes the HTR1A and RNF180 genes. This is the first report of an individual with an apparently balanced complex chromosomal rearrangement in conjunction with a microdeletion at 5q12.1–5q12.3 in which there are both mental-motor retardation and dysmorphia.     

Isoniazid-induced cell death is precipitated by underlying mitochondrial complex I dysfunction in mouse hepatocytes

Isoniazid (INH) is an antituberculosis drug that has been associated with idiosyncratic liver injury in susceptible patients. The underlying mechanisms are still unclear, but there is growing evidence that INH and/or its major metabolite, hydrazine, may interfere with mitochondrial function. However, hepatic mitochondria have a large reserve capacity, and minor disruption of energy homeostasis does not necessarily induce cell death. We explored whether pharmacologic or genetic impairment of mitochondrial complex I may amplify mitochondrial dysfunction and precipitate INH-induced hepatocellular injury. We found that INH (≤3000 μM) did not induce cell injury in cultured mouse hepatocytes, although it decreased hepatocellular respiration and ATP levels in a concentration-dependent fashion. However, coexposure of hepatocytes to INH and nontoxic concentrations of the complex I inhibitors rotenone (3 μM) or piericidin A (30 nM) resulted in massive ATP depletion and cell death. Although both rotenone and piericidin A increased MitoSox-reactive fluorescence, Mito-TEMPO or N-acetylcysteine did not attenuate the extent of cytotoxicity. However, preincubation of cells with the acylamidase inhibitor bis-p-nitrophenol phosphate provided protection from hepatocyte injury induced by rotenone/INH (but not rotenone/hydrazine), suggesting that hydrazine was the cell-damaging species. Indeed, we found that hydrazine directly inhibited the activity of solubilized complex II. Hepatocytes isolated from mutant Ndufs4^+/− mice, although featuring moderately lower protein expression levels of this complex I subunit in liver mitochondria, exhibited unchanged hepatic complex I activity and were therefore not sensitized to INH. These data indicate that underlying inhibition of complex I, which alone is not acutely toxic, can trigger INH-induced hepatocellular injury.     

Community Structure and Role Analysis in Biological Networks

Abstract

Complex network analysis has received increasing interest in recent years, which provides a remarkable tool to describe complex systems of interacting entities, particular for biological systems. In this paper, we propose a methodology for identifying the significant nodes of the networks, including core nodes, bridge nodes and high-influential nodes, based on the idea of community and two new ranking measures, InterRank and IntraRank. The results show the significant nodes form a small number in biological networks, and uncover the relative small number of which has advantage for reducing the dimensions of the network and possibly help to define new biological targets.     

The intrinsic stability of the human prion β-sheet region investigated by molecular dynamics

Human prion diseases are neurodegenerative disorders associated to the misfolding of the prion protein (PrP). Common features of prion disorders are the fibrillar amyloid deposits and the formation of prefibrillar oligomeric species also suggested as the origin of cytotoxicity associated with diseases. Although the process of PrP misfolding has been extensively investigated, many crucial aspects of this process remain unclear. We have here carried out a molecular dynamics study to evaluate the intrinsic dynamics of PrP β-sheet, a region that is believed to play a crucial role in prion aggregation. Moreover, as this region mediates protein association in dimeric assemblies frequently observed in prion crystallographic investigations, we also analyzed the dynamics of these intermolecular interactions. The extensive sampling of replica exchange shows that the native antiparallel β-structure of the prion is endowed with a remarkable stability. Therefore, upon unfolding, the persistence of a structured β-region may seed molecular association and influence the subsequent phases of the aggregation process. The analysis of the four-stranded β-sheet detected in the dimeric assemblies of PrP shows a tendency of this region to form dynamical structured states. The impact on the β-sheet structure and dynamics of disease associated point mutations has also been evaluated.     

Life time—circadian clocks,mitochondria and metabolism

A functional circadian clock has long been considered a selective advantage. Accumulating evidence shows that the clock coordinates a variety of physiological processes in order to schedule them to the optimal time of day and thus to synchronize metabolism to changes in external conditions. In mitochondria, both metabolic and cellular defense mechanisms are carefully regulated. Abnormal clock function, might influence mitochondrial function, resulting in decreased fitness of an organism.     

Regularity of Daily Activities in Stroke

Various studies have been performed using the Social Rhythm Metric (SRM), though none has been developed with stroke patients. Stroke is a pathology that provokes a strong physical and social impact caused by an abnormality in cerebral circulation. Consequently, we performed two studies to validate the SRM and translate it into Portuguese, and to evaluate the regularity of the daily activities of stroke patients. Both healthy individuals and patients with unilateral cerebral lesions were evaluated. Subjects were of both sexes and between 45 and 65 yrs of age. Participants underwent clinical evaluation and recorded the time of 17 daily activities on the SRM for two weeks. Data were analyzed by the Pearson correlation and Fisher tests. After conceptual translation into Portuguese, corrections were made to arrive at the final version. Normative SRM scores varied from 3.2 to 7.0, suggesting that the activities presented in SRM adequately represented the daily routines of the patients. A correlation was found in SRM between the weeks (r=0.84; p=0.0001), indicating instrument reliability. The mean (±SD) score of the stoke patients was 4.8 (±1.0), and the correlation between the SRM and level of neurological damage showed that patients with lower SRM values were more physically compromised (r=?0.29; p=0.04), suggesting that SRM may be a clinical predictor. Activities related to eating and the sleep‐wake cycle were rated by most patients. In all, 71% of the patients did not work, while 84% of healthy individuals did (p=0.001). Only 64% of patients left home compared to 90% of the healthy subjects (p=0.001), and 59% of patients recorded the activity of going home compared to 82% of healthy individuals (p=0.001). According to the results, there is evidence of the validity and reliability of the SRM, enabling it to be reliably used in chronobiological studies of stroke patients. Given that a less regular lifestyle may be associated with neurological compromise and a decrease in social activities, we suggest new studies with the repeated application of this instrument over the clinical evolution of the disease to better define improvement or worsening of the patient's condition in terms of their social and health aspects.     

Glia Maturation Factor (GMF) Interacts with Arp2/3 Complex in a Nucleotide State-dependent Manner

Glia maturation factor (GMF) is a member of the actin-depolymerizing factor (ADF)/cofilin family. ADF/cofilin promotes disassembly of aged actin filaments, whereas GMF interacts specifically with Arp2/3 complex at branch junctions and promotes debranching. A distinguishing feature of ADF/cofilin is that it binds tighter to ADP-bound than to ATP-bound monomeric or filamentous actin. The interaction is also regulated by phosphorylation at Ser-3 of mammalian cofilin, which inhibits binding to actin. However, it is unknown whether these two factors play a role in the interaction of GMF with Arp2/3 complex. Here we show using isothermal titration calorimetry that mammalian GMF has very low affinity for ATP-bound Arp2/3 complex but binds ADP-bound Arp2/3 complex with 0.7 μm affinity. The phosphomimetic mutation S2E in GMF inhibits this interaction. GMF does not bind monomeric ATP- or ADP-actin, confirming its specificity for Arp2/3 complex. We further show that mammalian Arp2/3 complex nucleation activated by the WCA region of the nucleation-promoting factor N-WASP is not affected by GMF, whereas nucleation activated by the WCA region of WAVE2 is slightly inhibited at high GMF concentrations. Together, the results suggest that GMF functions by a mechanism similar to that of other ADF/cofilin family members, displaying a preference for ADP-Arp2/3 complex and undergoing inhibition by phosphorylation of a serine residue near the N terminus. Arp2/3 complex nucleation occurs in the ATP state, and nucleotide hydrolysis promotes debranching, suggesting that the higher affinity of GMF for ADP-Arp2/3 complex plays a physiological role by promoting debranching of aged branch junctions without interfering with Arp2/3 complex nucleation.     

Atypical Cohesin-Dockerin Complex Responsible for Cell Surface Attachment of Cellulosomal Components: BINDING FIDELITY,PROMISCUITY, AND STRUCTURAL BUTTRESSES*

The rumen bacterium Ruminococcus flavefaciens produces a highly organized multienzyme cellulosome complex that plays a key role in the degradation of plant cell wall polysaccharides, notably cellulose. The R. flavefaciens cellulosomal system is anchored to the bacterial cell wall through a relatively small ScaE scaffoldin subunit, which bears a single type IIIe cohesin responsible for the attachment of two major dockerin-containing scaffoldin proteins, ScaB and the cellulose-binding protein CttA. Although ScaB recruits the catalytic machinery onto the complex, CttA mediates attachment of the bacterial substrate via its two putative carbohydrate-binding modules. In an effort to understand the structural basis for assembly and cell surface attachment of the cellulosome in R. flavefaciens, we determined the crystal structure of the high affinity complex (K_d = 20.83 nm) between the cohesin module of ScaE (CohE) and its cognate X-dockerin (XDoc) modular dyad from CttA at 1.97-Å resolution. The structure reveals an atypical calcium-binding loop containing a 13-residue insert. The results further pinpoint two charged specificity-related residues on the surface of the cohesin module that are responsible for specific versus promiscuous cross-strain binding of the dockerin module. In addition, a combined functional role for the three enigmatic dockerin inserts was established whereby these extraneous segments serve as structural buttresses that reinforce the stalklike conformation of the X-module, thus segregating its tethered complement of cellulosomal components from the cell surface. The novel structure of the RfCohE-XDoc complex sheds light on divergent dockerin structure and function and provides insight into the specificity features of the type IIIe cohesin-dockerin interaction.     

Structure of the Type VI Effector-Immunity Complex (Tae4-Tai4) Provides Novel Insights into the Inhibition Mechanism of the Effector by Its Immunity Protein

The type VI secretion system (T6SS), a multisubunit needle-like apparatus, has recently been found to play a role in interspecies interactions. The Gram-negative bacteria harboring T6SS (donor) deliver the effectors into their neighboring cells (recipient) to kill them. Meanwhile, the cognate immunity proteins were employed to protect the donor cells against the toxic effectors. Tae4 (type VI amidase effector 4) and Tai4 (type VI amidase immunity 4) are newly identified T6SS effector-immunity pairs. Here, we report the crystal structures of Tae4 from Enterobacter cloacae and Tae4-Tai4 complexes from both E. cloacae and Salmonella typhimurium. Tae4 acts as a dl-endopeptidase and displays a typical N1pC/P60 domain. Unlike Tsi1 (type VI secretion immunity 1), Tai4 is an all-helical protein and forms a dimer in solution. The small angle x-ray scattering study combined with the analytical ultracentrifugation reveal that the Tae4-Tai4 complex is a compact heterotetramer that consists of a Tai4 dimer and two Tae4 molecules in solution. Structure-based mutational analysis of the Tae4-Tai4 interface shows that a helix (α3) of one subunit in dimeric Tai4 plays a major role in binding of Tae4, whereas a protruding loop (L4) in the other subunit is mainly responsible for inhibiting Tae4 activity. The inhibition process requires collaboration between the Tai4 dimer. These results reveal a novel and unique inhibition mechanism in effector-immunity pairs and suggest a new strategy to develop antipathogen drugs.