Accumulation of Species-Specific Amino Acid Replacements That Cause Loss of Particular Protein Functions in Buchnera, an Endocellular Bacterial Symbiont

Endosymbiotic bacteria live in animal cells and are transmitted vertically at the time of the host's reproduction. In view of their small and asexual populations with infrequent chances of recombination, these endocellular bacteria are expected to accumulate mildly deleterious mutations. Previous studies showed that the DNA sequences of these bacteria evolved faster than those of free-living bacteria. In this study, we compared all the ORFs of Buchnera, an endocellular bacterial symbiont of aphids, with those of 34 other prokaryotic organisms and estimated the effect of the accelerated evolution of Buchnera on the functions of its proteins. It was revealed that Buchnera proteins contain many mutations at the sites where sequences are conserved in their orthologues in many other organisms. In addition, amino acid replacements at the conserved sites are mostly changes to physicochemically different amino acids. These results suggest that functions and conformations of Buchnera proteins have been seriously impaired or strongly modified. Indeed, extensive loss of functional motifs was observed in some Buchnera proteins. In many Buchnera proteins mutations were not detected evenly throughout each molecule but tended to accumulate in some functional units, possibly leading to loss of specific functions. As Buchnera has an unusual and limited gene repertory, it is conceivable that the manner of interactions among its proteins has been changed, and thus, functional constraints over their amino acid residues have also been changed during evolution. This may account for the loss of some functional units only in the Buchnera proteins. We obtained evidence that amino acid replacements in Buchnera were not always deleterious, but neutral or, in some cases, even positively selected. Received: 14 December 2000 / Accepted: 12 March 2001     

Four Rice Seed cDNA Clones Belonging to the α-Amylase/Trypsin Inhibitor Gene Family Encode Potential Rice Allergens

Four rice seed proteins encoded by cDNAs belonging to the α-amylase/trypsin inhibitor gene family were overexpressed as TrpE-fusion proteins in E. coli. The expressed rice proteins were detected by SDS-PAGE as major proteins in bacterial celllysates. Western blot analyses showed that all the recombinant proteins were immunologically reactive to rabbit polyclonal antibodies and to a mouse monoclonal antibody (25B9) specific for a previously isolated rice allergen of 16 kDa. Some truncated proteins from deletion mutants of the cDNAs retained their reactivity to the specific antibodies. These results suggest that the cDNAs encode potential rice allergens and that some epitopes of the recombinant proteins are still immunoreactive when they are expressed as their fragments.     

In-vitro dual binding activity of a evolutionarily related subgroup of hnRNP proteins

The wide family of heterogeneous nuclear ribonucleoproteins (hnRNPs) comprises members that interact with single-stranded nucleic acids. On the basis of their structure, some of them are characterised by a tandem RNA-binding domain (RBD) and a glycine-rich C-terminus, showing a high degree of homology. Recently, we have isolated some proteins belonging to this group that interact with single-stranded cytosine-block telomeric DNA. The aim of the present investigation is to better characterise the relationship of some structural features shared by these proteins and their in-vitro interaction with the telomeric type sequences. We analysed the in-vitro binding properties of some of these components toward both single-stranded telomeric motifs. Using deletion mutants, the relationship between cytosine-rich motif binding activity and the structural features of one of these proteins is further characterized. This binding activity appears to be related to a subgroup of the 2xRBD+Glycine rich hnRNP, suggesting functionally distinct properties of these proteins, in agreement with their evolutionary relationship. (Mol Cell Biochem 268: 121–127, 2005)     

哺乳动物中Kelch超家族蛋白的结构与功能

在不同生物体,如病毒、植物、真菌和哺乳动物中,几百种含Kelch构象的蛋白质已经被鉴定出来了,但大部分此类蛋白质的功能未知.而在哺乳动物中,已经有接近41种Kelch蛋白被报道.这些蛋白质在体内参与了很多重要的生理过程.根据Kelch蛋白的域结构不同,把这些哺乳动物中的Kelch蛋白分成了三类(BTB/Kelch蛋白,只含有Kelch结构域的蛋白质,及含其他结构域的Kelch蛋白),并把其已知的功能大致划分为5类:介导蛋白质-蛋白质的相互作用、参与蛋白质降解、信号传导、胞外功能及其他等.另外还认为Keap 1的Kelch结构域其晶体结构应该是研究哺乳动物Kelch蛋白的一个很好的参考模型.     

Quantitative chemical proteomics profiling of de novo protein synthesis during starvation-mediated autophagy

Autophagy is an intracellular degradation mechanism in response to nutrient starvation. Via autophagy, some nonessential cellular constituents are degraded in a lysosome-dependent manner to generate biomolecules that can be utilized for maintaining the metabolic homeostasis. Although it is known that under starvation the global protein synthesis is significantly reduced mainly due to suppression of MTOR (mechanistic target of rapamycin serine/threonine kinase), emerging evidence demonstrates that de novo protein synthesis is involved in the autophagic process. However, characterizing these de novo proteins has been an issue with current techniques. Here, we developed a novel method to identify newly synthesized proteins during starvation-mediated autophagy by combining bio-orthogonal noncanonical amino acid tagging (BONCAT) and isobaric tags for relative and absolute quantitation (iTRAQ^TM). Using bio-orthogonal metabolic tagging, L-azidohomoalanine (AHA) was incorporated into newly synthesized proteins which were then enriched with avidin beads after a click reaction between alkyne-bearing biotin and AHA's bio-orthogonal azide moiety. The enriched proteins were subjected to iTRAQ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Via the above approach, we identified and quantified a total of 1176 proteins and among them 711 proteins were found to meet our defined criteria as de novo synthesized proteins during starvation-mediated autophagy. The characterized functional profiles of the 711 newly synthesized proteins by bioinformatics analysis suggest their roles in ensuring the prosurvival outcome of autophagy. Finally, we performed validation assays for some selected proteins and found that knockdown of some genes has a significant impact on starvation-induced autophagy. Thus, we think that the BONCAT-iTRAQ approach is effective in the identification of newly synthesized proteins and provides useful insights to the molecular mechanisms and biological functions of autophagy.     

Illuminating how malaria parasites export proteins into host erythrocytes

Plasmodium parasites that cause the disease malaria have developed an elaborate trafficking pathway to facilitate the export of hundreds of effector proteins into their host cell, the erythrocyte. In this review, we outline how certain effector proteins contribute to parasite survival, virulence, and immune evasion. We also highlight how parasite proteins destined for export are recognised at the endoplasmic reticulum to facilitate entry into the export pathway and how the effector proteins are able to transverse the bounding parasitophorous vaculoar membrane via the Plasmodium translocon of exported proteins to gain access to the host cell. Some of the gaps in our understanding of the export pathway are also presented. Finally, we examine the degree of conservation of some of the key components of the Plasmodium export pathway in closely related apicomplexan parasites, which may provide insight into how the diverse apicomplexan parasites have adapted to survival pressures encountered within their respective host cells.     

Biochemical Studies on Indian Wild Legumes

Chemical screening of some profusely growing wild leguminous seeds of Acacia, Albizzia and Leucaena species, for their nutritional constituents suggested the possibility of their inclusion in animal nutrition. However, since the whole seeds proved to be unpalatable as well as toxic in some cases, it was thought of utility to employ their isolated proteins instead. Accordingly, with a view to explore newer and effective sources of dietary proteins, simple and effective solubility method was employed for their extraction, precipitation, fractionation and isolation from the undesirable wild seed constituents to obtain the protein fractions in the pure form which could be incorporated in animal nutrition. Distribution of total nitrogen and dispersion of nitrogenous constituents in the seeds have also been studied. Extraction of proteins of considerable purity is best effected with sodium chloride solution (5%, w/v) and subsequent dialysis of the extract. Proteins were isolated from seven varieties of leguminous seeds by acid-alkali extraction method and their protein efficiency ratio and the biological value have been evaluated by animal experiments with and without supplementing the isolated protein diets with the missing essential amino acids.     

A Two-Dimensional Separation of Proteins from Chloroplast Thylakoids and Other Membranes

• 1 A two-dimensional, highly reproducible, analytical separation procedure for Triton/urea extractable proteins, using isoelectric focusing in the first dimension and gelelectrophoresis with sodium dodecylsulfate (SDS) in the second, is described. The separation of erythrocyte membrane proteins compares the reliability with that of published procedures.
• 2 From the thylakoid membranes of the green alga Chlamydomonas reinhardii and of spinach, two-dimensional maps of the Triton/urea extractable proteins were established, containing 58 and 33 peptide spots, respectively, which were characterized by their molecular weights and apparent isoelectric points. In Chlamydomonas some of the spots could be attributed to the photosystem I and II particles. A few major spots in the maps of the thylakoid proteins from the two non-related plants were found at the same relative positions.
• 3 A one-dimensional electrophoretic comparison of the SDS and the Triton/urea-extractable proteins showed that in the Triton/urea extract of thylakoid membranes some proteins of the photosystem II region, and in that of erythrocyte membranes, some components of the band 3 and 5 region are reduced or missing.

     

Role of small RNAs in the interaction between Arabidopsis and Pseudomonas syringae

Endogenous small RNAs (miRNAs and siRNAs) regulate gene expression in diverse biological processes.Research with the Arabidopsis-Pseudomonas syringae system has shown that small RNAs contribute to plan...     

The Contribution of Peripheral Stem-Loops to the Catalytic Activity of Archaeal RNase P RNA from Pyrococcus horikoshii OT3

We investigated the contribution of peripheral stem-loops to the catalytic activity of an archaeal RNase P RNA, PhopRNA, from Pyrococcus horikoshii OT3. PhopRNA mutants, in which the stem-loops were individually deleted, were prepared and characterized with respect to precursor tRNA (pre-tRNA) cleavage activity in the presence of five RNase P proteins. All the mutants retained the activity to some extent, indicating that they are moderately implicated in catalysis. Further characterization suggested that the stem-loops serve largely as binding sites for the proteins, and that their interactions are predominantly involved in stabilization of the active conformation of PhopRNA.     

DNA-Binding Proteins and Structural Characteristics of Chloroplast Nucleoids

A comparative analysis of proteins from chloroplast nucleoids was performed in two higher-plant species (Pisum sativumL. andArabidopsis thalianaL.) and a green alga Chlamydomonas reinhardtiiDang. In the nucleoids of the higher plants and the alga, 26–27 proteins were detected with their mol wts ranging from 10 to more than 94 kD. In all the species tested, the distribution of nucleoid proteins by their mol wts was similar, especially between the predominant proteins with mol wts of 10 to 40 kD. Six DNA-binding proteins (12–18 kD) were detected in nucleoids fromCh. reinhardtiichloroplasts after in vivocovalent cross-linking between chloroplast DNA and proteins. Under an electron miscroscope, some regular structures resembling nucleosome-like particles of bacterial cells were revealed.     

Proteomic analysis of residual proteins in blades and petioles of fallen leaves of Brassica napus

Brassica napus L. is an important crop plant, characterised by high nitrogen (N) levels in fallen leaves, leading to a significant restitution of this element to the soil, with important consequences at the economic and environmental levels. It is now well established that the N in fallen leaves is due to weak N remobilisation that is especially related to incomplete degradation of foliar proteins during leaf senescence. Identification of residual proteins in a fallen leaf (i.e. incompletely degraded in the last step of the N remobilisation process) constitutes important information for improving nutrient use efficiency. Proteome analysis of the vascular system (petioles) and blades from fallen leaves of Brassica napus was performed, and the 30 most abundant residual proteins in each tissue were identified. Among them, several proteins involved in N recycling remain in the leaf after abscission. Moreover, this study reveals that some residual proteins are associated with energy metabolism, protection against oxidative stress, and more surprisingly, photosynthesis. Finally, comparison of blade and petiole proteomes show that, despite their different physiological roles in the non‐senescing leaf, both organs redirect their metabolism in order to ensure catabolic reactions. Taken together, the results suggest that a better degradation of these leaf proteins during the senescence process could enable improvements in the N use efficiency of Brassica napus.     

Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery

Mycobacterium tuberculosis encodes mycobactin, a peptide siderophore that is biosynthesized by a nonribosomal peptide synthetase (NRPS) mechanism. Within the mycobactin biosynthetic gene cluster is a gene that encodes a 71-amino-acid protein MbtH. Many other NRPS gene clusters harbor mbtH homologs, and recent genetic, biochemical, and structural studies have begun to shed light on the function(s) of these proteins. In some cases, MbtH-like proteins are required for biosynthesis of their cognate peptides, and non-cognate MbtH-like proteins have been shown to be partially complementary. Biochemical studies revealed that certain MbtH-like proteins participate in tight binding to NRPS proteins containing adenylation (A) domains where they stimulate adenylation reactions. Expression of MbtH-like proteins is important for a number of applications, including optimal production of native and genetically engineered secondary metabolites produced by mechanisms that employ NRPS enzymes. They also may serve as beacons to identify gifted actinomycetes and possibly other bacteria that encode multiple functional NRPS pathways for discovery of novel secondary metabolites by genome mining.     

Removal of Surface Glycoproteins and Transfer among <Emphasis Type="Italic">Brachionus</Emphasis> species

Glycoproteins on the body surface of females of the rotifer Brachionus plicatilis are a key signal in their mate recognition system. When B. plicatilis Russian strain females were exposed to 50 mM EDTA or EGTA, several surface glycoproteins were removed. Females exposed to EDTA died, but remained intact and were used in mating bioassays with conspecifics males. Live control females elicited a male mating response in 21% of encounters, freeze-killed control females elicited responses in 23%, but EDTA extracted females elicited a mating response in only 5% of encounters. At least some of the EDTA-extractable proteins on the surface of females appear to be critical to male mate recognition. EDTA treated females could be exposed to proteins extracted from other females and some proteins re-attached to their body surface, restoring their attractiveness to males. SDS-PAGE of these proteins revealed 15–17 prominent bands, most ranging in molecular mass from 66 to 12 kD. The EDTA-extractable proteins were separated using ion exchange chromatography and each fraction was tested for its ability to restore female attractiveness. When proteins in fraction 22 were bound to females, they restored 80% of the females’ ability to elicit male mating responses. Exposing EDTA treated females to bovine serum albumin or casein had no effect on their attractiveness to males. EDTA treated females from different Brachionus clades and species were exposed to proteins from fraction 22. Female attractiveness could be restored in most clades of B. plicatilis, but no transfer of mating attractiveness was observed to B. rotundiformis or B. ibericus females. Conspecific males treated with EDTA and exposed to proteins in fraction 22 could not be feminized and made attractive to other males. A sexual dimorphism in surface proteins therefore exists between B. plicatilis females and males. Successful transfer of glycoproteins critical in mate recognition is dependent on signal glycoprotein structure and the structure of the other proteins present on the surface of females.     

Heme proteins—Diversity in structural characteristics,function, and folding

The characteristics of heme prosthetic groups and their binding sites have been analyzed in detail in a data set of nonhomologous heme proteins. Variations in the shape, volume, and chemical composition of the binding site, in the mode of heme binding and in the number and nature of heme–protein interactions are found to result in significantly different heme environments in proteins with different functions in biology. Differences are also seen in the properties of the apo states of the proteins. The apo states of proteins that bind heme permanently in their functional form show some disorder, ranging from local unfolding in the heme binding pocket to complete unfolding to give a random coil. In contrast, proteins that bind heme transiently are fully folded in their apo and holo states, presumably allowing both apo and holo forms to remain biologically active resisting aggregation or proteolysis. The principles identified here provide a framework for the design of de novo proteins that will exhibit tight heme ligand binding and for the identification of the function of structural genomic target proteins with heme ligands. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Assembly,sorting, and exit of oligomeric proteins from the endoplasmic reticulum

The endoplasmic reticulum (ER) uses various mechanisms to ensure that only properly folded proteins enter the secretory pathway. For proteins that oligomerize in the ER, the proper tertiary and quaternary structures must be achieved before their release. Although some proteins fold before oligomerization, others initiate oligomerization cotranslationally. Here, we discuss these different strategies and some of the unique problems they present for the ER quality control system. One mechanism used by the ER is thiol retention. Thiol retention operates by monitoring the redox state of specific cysteine residue(s) and was discovered in studies on the assembly of IgM, a complex oligomeric glycoprotein. This system is also involved in retaining other unassembled proteins in the ER. Mutations that result in uneven numbers of cysteine residues can subject yet other proteins to thiol retention, altering their oligomerization status and function. The implications of these results on the effects of thiol retention on protein function and cell fate are discussed. BioEssays 20 :546–554, 1998. © 1998 John Wiley & Sons Inc.