Mitochondrial Tim9 protects Tim10 from degradation by the protease Yme1

Translocase of IM (inner membrane; Tim)9 and Tim10 are essential homologue proteins of the mitochondrial intermembrane space (IMS) and form a stable hexameric Tim9–Tim10 complex there. Redox-switch of the four conserved cysteine residues plays a key role during the biogenesis of these proteins and, in turn, the Tim proteins play a vital chaperone-like role during import of mitochondrial membrane proteins. However, the functional mechanism of the small Tim chaperones is far from solved and it is unclear whether the individual proteins play specific roles or the complex functions as a single unit. In the present study, we examined the requirement and role for the individual disulfide bonds of Tim9 on cell viability, complex formation and stability using yeast genetic, biochemical and biophysical methods. Loss of the Tim9 inner disulfide bond led to a temperature-sensitive phenotype and degradation of both Tim9 and Tim10. The growth phenotype could be suppressed by deletion of the mitochondrial i-AAA (ATPases associated with diverse cellular activities) protease Yme1, and this correlates strongly with stabilization of the Tim10 protein regardless of Tim9 levels. Formation of both disulfide bonds is not essential for Tim9 function, but it can facilitate the formation and improve the stability of the hexameric Tim9–Tim10 complex. Furthermore, our results suggest that the primary function of Tim9 is to protect Tim10 from degradation by Yme1 via assembly into the Tim9–Tim10 complex. We propose that Tim10, rather than the hexameric Tim9–Tim10 complex, is the functional form of these proteins.     

烟粉虱及温室白粉虱为害后对后续粉虱种群的影响

【目的】分析烟粉虱Bemisia tabaci(Gennadius)或温室白粉虱Trialeurodes vaporariorum(Westwood)为害番茄后对后续温室白粉虱和烟粉虱生长、发育、成虫寿命和繁殖等产生的促进或抑制作用,可为明确寄主植物番茄介导的温室白粉虱-烟粉虱的种间互作,开展粉虱的科学防控提供科学依据。【方法】通过在寄主植物番茄叶片上先将B型烟粉虱或温室白粉虱按不同的顺序、时间间隔分开接种,再系统观察后续接上的两种粉虱生长、发育、繁殖等种群参数的变化。【结果】(1)先期接上烟粉虱对后续温室白粉虱的发育、寿命、产卵量有明显的促进作用;这种作用需烟粉虱的持续诱导,若去掉烟粉虱,其对温室白粉虱的促进作用即消失;先期接上温室白粉虱可缩短后续烟粉虱伪蛹期,但温室白粉虱的持续存在不利于烟粉虱的产卵,且明显降低烟粉虱的内禀增长率和净增殖率。(2)先后在番茄上同时接上温室白粉虱可降低后续烟粉虱的单雌产卵量和雌、雄虫的成虫寿命;先后同时接种烟粉虱却显著地增加了温室白粉虱的单雌产卵量;烟粉虱的提前存在降低了后续烟粉虱单雌产卵量。(3)但两种粉虱之间作用存在着一定的时间滞后性。烟粉虱对温室白粉虱的发育、寿命、产卵量产生的促进作用大约在其卵期后的一段时间才能显现出来;温室白粉虱对烟粉虱伪蛹期的缩短作用也需提前诱导;而温室白粉虱对温室白粉虱的促进作用相当滞后,直到成虫期才显现出来。【结论】B型烟粉虱和温室白粉虱之间可通过寄主番茄产生相互影响,前期烟粉虱为害可显著促进后续温室白粉虱卵和若虫的发育,而前期温室白粉虱为害对烟粉虱的发育不利。     

Key mutations stabilize antigen‐binding conformation during affinity maturation of a broadly neutralizing influenza antibody lineage

Affinity maturation, the process in which somatic hypermutation and positive selection generate antibodies with increasing affinity for an antigen, is pivotal in acquired humoral immunity. We have studied the mechanism of affinity gain in a human B‐cell lineage in which two main maturation pathways, diverging from a common ancestor, lead to three mature antibodies that neutralize a broad range of H1 influenza viruses. Previous work showed that increased affinity in the mature antibodies derives primarily from stabilization of the CDR H3 loop in the antigen‐binding conformation. We have now used molecular dynamics simulations and existing crystal structures to identify potentially key maturation mutations, and we have characterized their effects on the CDR H3 loop and on antigen binding using further simulations and experimental affinity measurements, respectively. In the two maturation pathways, different contacts between light and heavy chains stabilize the CDR H3 loop. As few as two single‐site mutations in each pathway can confer substantial loop stability, but none of them confers experimentally detectable stability on its own. Our results support models of the germinal center reaction in which two or more mutations can occur without concomitant selection and show how divergent pathways have yielded functionally equivalent antibodies. Proteins 2014; 83:771–780. © 2014 The Authors. Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Inferring the microscopic surface energy of protein–protein interfaces from mutation data

Mutations at protein–protein recognition sites alter binding strength by altering the chemical nature of the interacting surfaces. We present a simple surface energy model, parameterized with empirical values, yielding mean energies of ?48 cal mol^?1 Å^?2 for interactions between hydrophobic surfaces, ?51 to ?80 cal mol^?1 Å^?2 for surfaces of complementary charge, and 66–83 cal mol^?1 Å^?2 for electrostatically repelling surfaces, relative to the aqueous phase. This places the mean energy of hydrophobic surface burial at ?24 cal mol^?1 Å^?2. Despite neglecting configurational entropy and intramolecular changes, the model correlates with empirical binding free energies of a functionally diverse set of rigid‐body interactions (r = 0.66). When used to rerank docking poses, it can place near‐native solutions in the top 10 for 37% of the complexes evaluated, and 82% in the top 100. The method shows that hydrophobic burial is the driving force for protein association, accounting for 50–95% of the cohesive energy. The model is available open‐source from http://life.bsc.es/pid/web/surface_energy/ and via the CCharpPPI web server http://life.bsc.es/pid/ccharppi/ . Proteins 2015; 83:640–650. © 2015 Wiley Periodicals, Inc.     

Effect of age,sex, and mutual interaction on the locomotor behavior of Orchestia gammarellus in the supralittoral zone of Ghar El Melh lagoon (Bizerte,Tunisia)

Freshly collected individuals of Orchestia gammarellus from the supralittoral zone of Ghar El Melh lagoon (Northern of Tunisia) were housed in spring in a controlled-environment cabinet. Locomotor activity rhythm of this species was recorded in spring, at a constant temperature of 18 ± 0.5 °C. In the first experiment, juveniles and adults specimens were kept under light-dark cycle in phase with the natural diel cycle. In the two other experiments (males/females and mixed/unmixed groups), individuals of O. gammarellus were maintained under constant darkness. According to double-plotted actograms, waveforms and periodogram analysis, results revealed different locomotor pattern. However, locomotor rhythm of juveniles was more stable than adults. Also, the locomotor activity rhythm of groups was more defined than that of the isolated individuals. Moreover, the activity of mixed groups as well as unmixed groups was more pronounced than the activity of the isolated individuals. Furthermore, results showed no significant difference between locomotor rhythm pattern of males and females of this species.     

Structural studies of the C‐terminal 19‐peptide of serum amyloid A and its Pro→Ala variants interacting with human cystatin C

Serum amyloid A (SAA) is a multifunctional acute‐phase protein whose concentration in serum increases markedly following a number of chronic inflammatory and neoplastic diseases. Prolonged high SAA level may give rise to reactive systemic amyloid A (AA) amyloidosis, where the N‐terminal segment of SAA is deposited as amyloid fibrils. Besides, recently, well‐documented association of SAA with high‐density lipoprotein or glycosaminoglycans, in particular heparin/heparin sulfate (HS), and specific interaction between SAA and human cystatin C (hCC), the ubiquitous inhibitor of cysteine proteases, was proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, a hCC binding site in the SAA sequence was determined as SAA(86–104). The role of this SAA C‐terminal fragment as a ligand‐binding locus is still not clear. It was postulated important in native SAA folding and in pathogenesis of AA amyloidosis. In the search of conformational details of this SAA fragment, we did its structure and affinity studies, including its selected double/triple Pro→Ala variants. Our results clearly show that the SAA(86–104) 19‐peptide has rather unordered structure with bends in its C‐terminal part, which is consistent with the previous results relating to the whole protein. The results of affinity chromatography, fluorescent ELISA‐like test, CD and NMR studies point to an importance of proline residues on structure of SAA(86–104). Conformational details of SAA fragment, responsible for hCC binding, may help to understand the objective of hCC–SAA complex formation and its importance for pathogenesis of reactive amyloid A amyloidosis. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of bottom trawling on fish foraging and feeding

The effects of bottom trawling on benthic invertebrates include reductions of biomass, diversity and body size. These changes may negatively affect prey availability for demersal fishes, potentially leading to reduced food intake, body condition and yield of fishes in chronically trawled areas. Here, the effect of trawling on the prey availability and diet of two commercially important flatfish species, plaice (Pleuronectes platessa) and dab (Limanda limanda), was investigated over a trawling intensity gradient in the Irish Sea. Previous work in this area has shown that trawling negatively affects the condition of plaice but not of dab. This study showed that reductions in local prey availability did not result in reduced feeding of fish. As trawling frequency increased, both fish and prey biomass declined, such that the ratio of fish to prey remained unchanged. Consequently, even at frequently trawled sites with low prey biomass, both plaice and dab maintained constant levels of stomach fullness and gut energy contents. However, dietary shifts in plaice towards energy-poor prey items were evident when prey species were analysed individually. This, together with a potential decrease in foraging efficiency due to low prey densities, was seen as the most plausible cause for the reduced body condition observed. Understanding the relationship between trawling, benthic impacts, fish foraging and resultant body condition is an important step in designing successful mitigation measures for future management strategies in bottom trawl fisheries.     

Intrinsic site‐selectivity of ubiquitin dimer formation

The post‐translational modification of proteins with ubiquitin can take on many forms, including the decoration of substrates with polymeric ubiquitin chains. These chains are linked through one of the seven lysine residues in ubiquitin, with the potential to form a panoply of linkage combinations as the chain length increases. The ensuing structural diversity of modifications serves a variety of signaling functions. Still, some linkages are present at a much higher level than others in cellulo. Although ubiquitination is an enzyme‐catalyzed process, the large disparity of abundancies led us to the hypothesis that some linkages might be intrinsically faster to form than others, perhaps directing the course of enzyme evolution. Herein, we assess the kinetics of ubiquitin dimer formation in an enzyme‐free system by measuring the rate constants for thiol–disulfide interchange between appropriate ubiquitin variants. Remarkably, we find that the kinetically expedient linkages correlate with those that are most abundant in cellulo. As the abundant linkages also appear to function more broadly in cellulo, this correlation suggests that the more accessible chains were selected for global roles.