Phylogenetic implications and diagenetic stability of macromolecules from pleistocene and recent shells of Mercenaria mercenaria (mollusca,bivalvia)

The phylogeny and diagenesis of Pleistocene and Recent bivalves were studied immunologically by use of a conventional antiserum elicited against an EDTA‐soluble macromolecular extract from shells of the modern bivalve mollusc Mercenaria mercenaria. ELISA tests of the antiserum with shell fragments of a wide range of modern bivalves gave taxonomically significant results. The antiserum reacted with palaeoheterodonts and heterodonts but not with representatives of other bivalve subclasses. This phylogenetic reactivity was also apparent in tests with fossil shells, although the specificity and overall strength of the reaction were both reduced. Absorption of the antiserum with etched shell powders of various (palaeo)heterodonts yielded more specific antibody preparations.

Investigations of shell matrix diagenesis, using the anti‐Mercenaria serum, demonstrated that small amounts of original determinants could be detected even in fossils over one million years old. The reactivity of the serum with extracts of fossil Mercenaria decreased with sample age. The relationship between serum reactivity and the degree of amino acid racemization was almost linear. Clearly, the various determinants to which antibodies were elicited were being destroyed at different rates.     

Calpain-mediated proteolysis of microtubule associated proteins MAP1B and MAP2 in developing brain

Microtubule associated proteins MAP1B and MAP2 are important components of the neuronal cytoskeleton. During early development of the brain, MAP1B (340 kDa) is present as two isoforms that differ in their level of phosphorylation, while MAP2 is expressed as a single high molecular weight isoform (MAP2B, 280 kDa) and a low molecular weight form (MAP2C, 70 kDa). In this study we examined and compared the sensitivities of MAP1B and MAP2, obtained from MT preparations and brain homogenates of young rats, to degradation by calcium-activated neutral protease, calpain II. We found that in MAPs prepared from microtubules the two isoforms of MAP1B had comparable sensitivity to calpain-mediated proteolysis. Similarly, the high and low molecular weight forms of MAP2 were equally sensitive to digestion by calpain. However, although both MAPs were very susceptible to calpain-mediated proteolysis, MAP1B was more resistant to degradation by calpain than MAP2. Furthermore, the endogenous degradation of MAPs in neonate brain homogenates was calcium-dependent and inhibited by leupeptin, and the pattern of degradation products for MAP1B and MAP2 was similar to that of calpain-mediated proteolysis. These data suggest that calpain can play a role in the regulation of MAPs levels during brain development, in relation to normal neuronal differentiation and disorders associated with neurodegeneration.     

Large-scale mapping of human protein interactome using structural complexes

Although the identification of protein interactions by high-throughput (HTP) methods progresses at a fast pace, 'interactome' data sets still suffer from high rates of false positives and low coverage. To map the human protein interactome, we describe a new framework that uses experimental evidence on structural complexes, the atomic details of binding interfaces and evolutionary conservation. The structurally inferred interaction network is highly modular and more functionally coherent compared with experimental interaction networks derived from multiple literature citations. Moreover, structurally inferred and high-confidence HTP networks complement each other well, allowing us to construct a merged network to generate testable hypotheses and provide valuable experimental leads.     

贝壳的有机质及生物矿化机制分析

以腹足纲贝壳香螺壳为研究对象,用弱酸去钙法进行蛋白提取,采用280纳米(A280)光吸收法测定蛋白含量,并通过聚丙烯酰胺(SDS-PAGE)凝胶电泳法对蛋白按照分子量大小的区别进行分离.实验结果表明香螺壳中蛋白含量和种类较少,其文石层比方解石层蛋白含量高的多,总量分别为0.89%和0.0533%;文石层分离出五种分子量的可溶性蛋白和四种分子量的不可溶性蛋白;而方解石层中分离出三种分子量的可溶性蛋白和三种分子量的不可溶性蛋白,且分子量不相同.正是这少量的蛋白质对贝壳的生物矿化过程和不同晶型的形成起着决定性作用.     

Variation among chicken stocks in the fractional rates of muscle protein synthesis and degradation

Fractional rates (% · day^–1) of synthesis and degradation were determined by measuring the output of N-methylhistidine (MeHis) in the excreta at 4 and 8 weeks of age in the chicken. At 4 weeks of age, the fractional rate of synthesis of the meat-type stock was twice that of the egg-type stock (White Leghorn), but the fractional rates of synthesis at 8 weeks of age were similar (4.1–5.1% · day^–1) among stocks. The fractional rate of degradation (1.3–1.5% · day^–1) of the meat-type stock at 8 weeks of age was less than half the rate of the egg-type stock (2.9% · day^–1). The fractional rates of synthesis and degradation at 4 weeks of age in the Satsuma native fowl were relatively high compared with those in the other stocks. In particular, the rate of degradation (8.6% · day^–1) at 4 weeks of age was approximately twice that of other stocks. These results show that fractional rates of synthesis and degradation of muscle protein in the chicken differ among genetically diverse groups. The effect of changes in rates of synthesis and degradation on the change in fractional growth rate also differed. From regression coefficients (b_{K s · FGR} and b_{K d · FGR}) of these rates in skeletal muscle protein on the fractional growth rate, it was recognized that the change in growth rate accompanies the changes in both synthesis and degradation in White Leghorn and commercial broilers but only the change in synthesis in White Plymouth Rock (dw) and Satsuma native fowl.     

On the molecular mechanisms of mitotic kinase activation

During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell''s shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.     

翡翠贻贝外套膜转录组及贝壳珍珠质层和肌棱柱层蛋白质组分析

贝类贝壳在生物材料学及仿生学研究中占据着重要地位。贝壳基质蛋白质是贝壳中的主要有机质成分,对贝壳的形成以及贝壳的力学性能至关重要。翡翠贻贝(Perna viridis)贝壳主要由肌棱柱层和珍珠质层两种微观结构组成,其结构层次较简单,是研究贝壳基质蛋白质及其与贝壳形成关系的极好材料。为深入研究翡翠贻贝贝壳基质蛋白质的分子组成以及分布特点,首先采用扫描电子显微镜,观察翡翠贻贝贝壳内表面珍珠质层和肌棱柱层的微观结构;采用刮取法获得贝壳内表面珍珠质层和肌棱柱层的粉末;对不同层次的贝壳粉末,利用酸溶法去除碳酸钙成分,所获得的有机质组分通过离心将其分为酸可溶性组分和酸不溶性组分。采用Illumina深度测序技术对翡翠贻贝外套膜组织进行大规模测序和序列组装,在此基础上,采用LC-MS/MS质谱技术结合外套膜转录组数据库搜索,对翡翠贻贝肌棱柱层和珍珠质层贝壳基质蛋白质开展组学分析。扫描电镜观察结果表明,翡翠贻贝贝壳有两种不同形貌结构的层次,其中珍珠质层为片状堆叠结构,而肌棱柱层为柱状结构。翡翠贻贝外套膜转录组测序共计获得 69 859 条Unigene。蛋白质组学鉴定结果表明,翡翠贻贝贝壳中总计鉴定到蛋白质54种,其中38种为肌棱柱层所特有蛋白质,3种珍珠质层特有蛋白质,另有13种在珍珠质层和肌棱柱层均被鉴定到。肌棱柱层特有蛋白质的分子多样性明显强于珍珠质层。上述研究为进一步探讨贝壳不同微观层次的形成机制,以及贝壳基质蛋白质对贝壳不同结构层次的调控作用机制奠定了基础。     

Flexibility of alpha-helices: results of a statistical analysis of database protein structures

Alpha-helices stand out as common and relatively invariant secondary structural elements of proteins. However, alpha-helices are not rigid bodies and their deformations can be significant in protein function (e.g. coiled coils). To quantify the flexibility of alpha-helices we have performed a structural principal-component analysis of helices of different lengths from a representative set of protein folds in the Protein Data Bank. We find three dominant modes of flexibility: two degenerate bend modes and one twist mode. The data are consistent with independent Gaussian distributions for each mode. The mode eigenvalues, which measure flexibility, follow simple scaling forms as a function of helix length. The dominant bend and twist modes and their harmonics are reproduced by a simple spring model, which incorporates hydrogen-bonding and excluded volume. As an application, we examine the amount of bend and twist in helices making up all coiled-coil proteins in SCOP. Incorporation of alpha-helix flexibility into structure refinement and design is discussed.     

Analysis of loop boundaries using different local structure assignment methods

Loops connect regular secondary structures. In many instances, they are known to play important biological roles. Analysis and prediction of loop conformations depend directly on the definition of repetitive structures. Nonetheless, the secondary structure assignment methods (SSAMs) often lead to divergent assignments. In this study, we analyzed, both structure and sequence point of views, how the divergence between different SSAMs affect boundary definitions of loops connecting regular secondary structures. The analysis of SSAMs underlines that no clear consensus between the different SSAMs can be easily found. Because these latter greatly influence the loop boundary definitions, important variations are indeed observed, that is, capping positions are shifted between different SSAMs. On the other hand, our results show that the sequence information in these capping regions are more stable than expected, and, classical and equivalent sequence patterns were found for most of the SSAMs. This is, to our knowledge, the most exhaustive survey in this field as (i) various databank have been used leading to similar results without implication of protein redundancy and (ii) the first time various SSAMs have been used. This work hence gives new insights into the difficult question of assignment of repetitive structures and addresses the issue of loop boundaries definition. Although SSAMs give very different local structure assignments capping sequence patterns remain efficiently stable.     

The Endosomal Protein-Sorting Receptor Sortilin Has a Role in Trafficking α-1 Antitrypsin

Up to 1 in 3000 individuals in the United States have α-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.     

蛋白质的选择性降解机制——2004年诺贝尔化学奖部分工作介绍

如何识别和选择性降解蛋白质是细胞生命过程中的重要环节.泛素-蛋白酶体需能降解途径的发现,揭示了蛋白质在细胞内选择性降解的普遍方式.对于需要清除的蛋白质,通过其赖氨酸残基侧链ε-氨基连接多聚泛素链（降解标签）,继而在蛋白酶体中被降解.这种选择性降解机制对于维持蛋白质在细胞内含量的动态平衡起到了关键性作用.