Folding into an autophagosome: ATG5 sheds light on how plants do it

Autophagosomes arise in yeast and animals from the sealing of a cup-shaped double-membrane precursor, the phagophore. The concerted action of about 30 evolutionarily conserved autophagy related (ATG) proteins lies at the core of this process. However, the mechanisms allowing phagophore generation and its differentiation into a sealed autophagosome are still not clear in detail, and very little is known in plants. This is due in part to the scarcity of structurally informative, real-time imaging data of ATG proteins at the phagophore site. Among these, the ATG5 complex directs anchoring of ATG8 to the phagophore, an event required for membrane expansion. Detailed real-time and 3D imaging of ATG5, ATG8, and an ER marker at the expanding phagophore allowed us to propose a model for autophagosome formation in plants. This model implies tight connections of the growing phagophore with the outer face of the cortical endoplasmic reticulum and prompts new questions on the mechanism of autophagosome biogenesis.     

Immunopathological Changes in the Brain of Immunosuppressed Mice Experimentally Infected with Toxocara canis

Toxocariasis is a soil-transmitted helminthozoonosis due to infection of humans by larvae of Toxocara canis. The disease could produce cognitive and behavioral disturbances especially in children. Meanwhile, in our modern era, the incidence of immunosuppression has been progressively increasing due to increased incidence of malignancy as well as increased use of immunosuppressive agents. The present study aimed at comparing some of the pathological and immunological alterations in the brain of normal and immunosuppressed mice experimentally infected with T. canis. Therefore, 180 Swiss albino mice were divided into 4 groups including normal (control) group, immunocompetent T. canis-infected group, immunosuppressed group (control), and immunosuppressed infected group. Infected mice were subjected to larval counts in the brain, and the brains from all mice were assessed for histopathological changes, astrogliosis, and IL-5 mRNA expression levels in brain tissues. The results showed that under immunosuppression, there were significant increase in brain larval counts, significant enhancement of reactive gliosis, and significant reduction in IL-5 mRNA expression. All these changes were maximal in the chronic stage of infection. In conclusion, the immunopathological alterations in the brains of infected animals were progressive over time, and were exaggerated under the effect of immunosuppression as did the intensity of cerebral infection.     

Analysis of the stability of multimeric proteins by effective DeltaG and effective m-values

Analyzing the stability of a multimeric protein is challenging because of the intrinsic difficulty in handling the mathematical model for the folded multimer-unfolded monomer equilibrium. To circumvent this problem, we introduce the concept of effective stability, DeltaGeff (= -RTlnKeff), where Keff is the equilibrium constant expressed in monomer units. Analysis of the denaturant effect on DeltaGeff gives new insight into the stability of multimeric proteins. When a multimeric protein is mostly folded, the dependence of effective stability on denaturant concentration (effective m-value) is simply the m-value of its monomeric unit. However, when the protein is mostly unfolded, its stability depends on denaturant concentration with the m-value of its multimeric form. We also find that the effective m-value at the Cm is a good approximation of the apparent m-value determined by fitting the equilibrium unfolding data from multimeric proteins with a two-state monomer model. Moreover, when the m-value of a monomeric unit is estimated from its size, the effective stability of a multimeric protein can be determined simply from Cm and this estimated m-value. These simple and intuitive approaches will allow a facile analysis of the stability of multimeric proteins. These analyses are also applicable for high-throughput analysis of protein stability on a proteomic scale.     

Interactions of a Pop5/Rpp1 heterodimer with the catalytic domain of RNase MRP

Ribonuclease (RNase) MRP is a multicomponent ribonucleoprotein complex closely related to RNase P. RNase MRP and eukaryotic RNase P share most of their protein components, as well as multiple features of their catalytic RNA moieties, but have distinct substrate specificities. While RNase P is practically universally found in all three domains of life, RNase MRP is essential in eukaryotes. The structural organizations of eukaryotic RNase P and RNase MRP are poorly understood. Here, we show that Pop5 and Rpp1, protein components found in both RNase P and RNase MRP, form a heterodimer that binds directly to the conserved area of the putative catalytic domain of RNase MRP RNA. The Pop5/Rpp1 binding site corresponds to the protein binding site in bacterial RNase P RNA. Structural and evolutionary roles of the Pop5/Rpp1 heterodimer in RNases P and MRP are discussed.     

Identification of antigenic proteins in Trichomonas vaginalis

Trichomoniasis is a sexually transmitted disease due to infection with Trichomonas vaginalis, and it can cause serious consequences for women's health. To study the virulence factors of this pathogen, T. vaginalis surface proteins were investigated using polyclonal antibodies specific to the membrane fractions of T. vaginalis. The T. vaginalis expression library was constructed by cloning the cDNA derived from mRNA of T. vaginalis into a phage λ Uni-ZAP XR vector, and then used for immunoscreening with the anti-membrane proteins of T. vaginalis antibodies. The immunoreactive proteins identified included adhesion protein AP65-1, α-actinin, kinesin-associated protein, teneurin, and 2 independent hypothetical proteins. Immunofluorescence assays showed that AP65-1, one of the identified immunogenic clones, is prevalent in the whole body of T. vaginalis. This study led us to identify T. vaginalis proteins which may stimulate immune responses by human cells.     

Mixed osmolytes: the degree to which one osmolyte affects the protein stabilizing ability of another

Mixtures of organic osmolytes occur in cells of many organisms, raising the question of whether their actions on protein stability are independent or synergistic. To investigate this question it is desirable to develop a system that permits evaluation of the effect of one osmolyte on the efficacy of another to either force-fold or denature a protein. A means of evaluating the efficacy of an osmolyte is provided by its m-value, an experimental quantity that measures the ability of the osmolyte to force a protein to unfold or fold. An experimental system is presented that enables evaluations of the m-values of osmolytes in the presence and absence of a second osmolyte. The experimental system involves use of a marginally stable protein in 10 mM buffer (pH 7, 200 mM salt, and 34 degrees C) that is at the midpoint of its native to denatured transition. These conditions enable determination of m-values for protecting and denaturing osmolytes in the presence and absence of a second osmolyte, permitting assessment of the extent to which the two osmolytes affect each other's efficacy. The two osmolytes investigated in this work are the denaturing osmolyte, urea, and the protecting osmolyte, sarcosine. Results show unequivocally that neither osmolyte alters the efficacy of the other in forcing the protein to fold or unfold-the osmolytes act independently on the protein despite their combined concentrations being in the multi-molar range. These osmolytes avoid altering one another's efficacy at these high concentrations because the number of osmolyte interaction sites on the protein is large and the binding constants are quite small. Consequently, the site occupancies are low enough in number that the two osmolytes neither compete nor cooperate in interacting with the protein.     

Biotransformation of Meloxicam by <Emphasis Type="Italic">Cunninghamella blakesleeana</Emphasis>: Significance of Carbon and Nitrogen Source

Influence of carbon and nitrogen source, on biotransformation of meloxicam was studied by employing Cunninghamella blakesleeana NCIM 687 with an aim to achieve maximum transformation of meloxicam and in search of new metabolites. The transformation was confirmed by HPLC and based on LC–MS–MS data and previous reports the metabolites were predicted as 5-hydroxymethyl meloxicam, 5-carboxy meloxicam and a novel metabolite. The quantification of metabolites was performed using HPLC peak areas. The results obtained indicate that glucose as carbon source, ammonium nitrate as nitrogen source, were found to be optimum for maximum transformation of meloxicam. The study suggests the significance of these factors in biotransformation of meloxicam using microbial cultures. The fermentation was scaled up to 1 l level.     

A scalable, GFP-based pipeline for membrane protein overexpression screening and purification

We describe a generic, GFP-based pipeline for membrane protein overexpression and purification in Escherichia coli. We exemplify the use of the pipeline by the identification and characterization of E. coli YedZ, a new, membrane-integral flavocytochrome. The approach is scalable and suitable for high-throughput applications. The GFP-based pipeline will facilitate the characterization of the E. coli membrane proteome and serves as an important reference for the characterization of other membrane proteomes.     

Identification of Immunodominant B-cell Epitope Regions of Reticulocyte Binding Proteins in Plasmodium vivax by Protein Microarray Based Immunoscreening

Plasmodium falciparum can invade all stages of red blood cells, while Plasmodium vivax can invade only reticulocytes. Although many P. vivax proteins have been discovered, their functions are largely unknown. Among them, P. vivax reticulocyte binding proteins (PvRBP1 and PvRBP2) recognize and bind to reticulocytes. Both proteins possess a C-terminal hydrophobic transmembrane domain, which drives adhesion to reticulocytes. PvRBP1 and PvRBP2 are large (> 326 kDa), which hinders identification of the functional domains. In this study, the complete genome information of the P. vivax RBP family was thoroughly analyzed using a prediction server with bioinformatics data to predict B-cell epitope domains. Eleven pvrbp family genes that included 2 pseudogenes and 9 full or partial length genes were selected and used to express recombinant proteins in a wheat germ cell-free system. The expressed proteins were used to evaluate the humoral immune response with vivax malaria patients and healthy individual serum samples by protein microarray. The recombinant fragments of 9 PvRBP proteins were successfully expressed; the soluble proteins ranged in molecular weight from 16 to 34 kDa. Evaluation of the humoral immune response to each recombinant PvRBP protein indicated a high antigenicity, with 38-88% sensitivity and 100% specificity. Of them, N-terminal parts of PvRBP2c (PVX_090325-1) and PvRBP2 like partial A (PVX_090330-1) elicited high antigenicity. In addition, the PvRBP2-like homologue B (PVX_116930) fragment was newly identified as high antigenicity and may be exploited as a potential antigenic candidate among the PvRBP family. The functional activity of the PvRBP family on merozoite invasion remains unknown.     

Differential physiological roles of ESCRT complexes in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

Endosomal sorting complex required for transport (ESCRT) complexes are involved in endosomal trafficking to the lysosome, cytokinesis, and viral budding. Extensive genetic, biochemical, and structural studies on the ESCRT system have been carried out in yeast and mammalian systems. However, the question of how the ESCRT system functions at the whole organism level has not been fully explored. In C. elegans, we performed RNAi experiments to knock-down gene expression of components of the ESCRT system and profiled their effects on protein degradation and endocytosis of YP170, a yolk protein. Targeted RNAi knock-down of ESCRT-I (tsg-101 and vps-28) and ESCRT-III (vps-24, and vps-32.2) components interfered with protein degradation while knock-down of ESCRT-II (vps-25 and vps-36) and ESCRT-III (vps-20 and vps-24) components hampered endocytosis. In contrast, the knockdown of vps-37, another ESCRT-I component, showed no defect in either YP170 uptake or degradation. Depletion of at least one component from each complex — ESCRT-0 (hgrs-1), ESCRT-I (tsg-101, vps-28, and vps-37), ESCRT-II (vps-36), ESCRT-III (vps-24), and Vps4 (vps-4) — resulted in abnormal distribution of embryos in the uterus of worms, possibly due to abnormal ovulation, fertilization, and egglaying. These results suggest differential physiological roles of ESCRT-0, -I, -II, and -III complexes in the context of the whole organism, C. elegans.     

Structure of the Yersinia enterocolitica type III secretion translocator chaperone SycD

Many Gram-negative bacteria use a type III secretion (T3S) system to directly inject effector molecules into eucaryotic cells in order to establish a symbiotic or pathogenic relationship with their host. The translocation of many T3S proteins requires specialized chaperones from the bacterial cytosol. SycD belongs to a class of T3S chaperones that assists the secretion of pore-forming translocators and, specifically chaperones the translocators YopB and YopD from enteropathogenic Yersinia enterocolitica. In addition, SycD is involved in the regulation of virulence factor biosynthesis and secretion. In this study, we present two crystal structures of Y. enterocolitica SycD at 1.95 and 2.6 Å resolution, the first experimental structures of a T3S class II chaperone specific for translocators. The fold of SycD is entirely α-helical and reveals three tetratricopeptide repeat-like motifs that had been predicted from amino acid sequence. In both structures, SycD forms dimers utilizing residues from the first tetratricopeptide repeat motif. Using site-directed mutagenesis and size exclusion chromatography, we verified that SycD forms head-to-head homodimers in solution. Although in both structures, dimerization largely depends on the same residues, the two assemblies represent alternative dimers that exhibit different monomer orientations and overall shape. In these two distinct head-to-head dimers, both the concave and the convex surface of each monomer are accessible for interactions with the SycD binding partners YopB and YopD. A SycD variant carrying two point mutations in the dimerization interface is properly folded but defective in dimerization. Expression of this stable SycD monomer in Yersinia does not rescue the phenotype of a sycD null mutant, suggesting a physiological relevance of the dimerization interface.     

Enterococcus faecalis phosphomevalonate kinase

The six enzymes of the mevalonate pathway of isopentenyl diphosphate biosynthesis represent potential for addressing a pressing human health concern, the development of antibiotics against resistant strains of the Gram-positive streptococci. We previously characterized the first four of the mevalonate pathway enzymes of Enterococcus faecalis, and here characterize the fifth, phosphomevalonate kinase (E.C. 2.7.4.2). E. faecalis genomic DNA and the polymerase chain reaction were used to clone DNA thought to encode phosphomevalonate kinase into pET28b(+). Double-stranded DNA sequencing verified the sequence of the recombinant gene. The encoded N-terminal hexahistidine-tagged protein was expressed in Escherichia coli with induction by isopropylthiogalactoside and purified by Ni(++) affinity chromatography, yield 20 mg protein per liter. Analysis of the purified protein by MALDI-TOF mass spectrometry established it as E. faecalis phosphomevalonate kinase. Analytical ultracentrifugation revealed that the kinase exists in solution primarily as a dimer. Assay for phosphomevalonate kinase activity used pyruvate kinase and lactate dehydrogenase to couple the formation of ADP to the oxidation of NADH. Optimal activity occurred at pH 8.0 and at 37 degrees C. The activation energy was approximately 5.6 kcal/mol. Activity with Mn(++), the preferred cation, was optimal at about 4 mM. Relative rates using different phosphoryl donors were 100 (ATP), 3.6 (GTP), 1.6 (TTP), and 0.4 (CTP). K(m) values were 0.17 mM for ATP and 0.19 mM for (R,S)-5-phosphomevalonate. The specific activity of the purified enzyme was 3.9 micromol substrate converted per minute per milligram protein. Applications to an immobilized enzyme bioreactor and to drug screening and design are discussed.     

Elastic behavior of RecA-DNA helical filaments

Escherichia coli RecA protein forms a right-handed helical filament with DNA molecules and has an ATP-dependent activity that exchanges homologous strands between single-stranded DNA (ssDNA) and duplex DNA. We show that the RecA-ssDNA filamentous complex is an elastic helical molecule whose length is controlled by the binding and release of nucleotide cofactors. RecA-ssDNA filaments were fluorescently labelled and attached to a glass surface inside a flow chamber. When the chamber solution was replaced by a buffer solution without nucleotide cofactors, the RecA-ssDNA filament rapidly contracted approximately 0.68-fold with partial filament dissociation. The contracted filament elongated up to 1.25-fold when a buffer solution containing ATPgammaS was injected, and elongated up to 1.17-fold when a buffer solution containing ATP or dATP was injected. This contraction-elongation behavior was able to be repeated by the successive injection of dATP and non-nucleotide buffers. We propose that this elastic motion couples to the elastic motion and/or the twisting rotation of DNA strands within the filament by adjusting their helical phases.     

Expression,Identification and Purification of Dictyostelium Acetoacetyl-CoA Thiolase Expressed in Escherichia coli

Acetoacetyl-CoA thiolase (AT) is an enzyme that catalyses the CoA-dependent thiolytic cleavage of acetoacetyl-CoA to yield 2 molecules of acetyl-CoA, or the reverse condensation reaction. A full-length cDNA clone pBSGT-3, which has homology to known thiolases, was isolated from Dictyostelium cDNA library. Expression of the protein encoded in pBSGT-3 in Escherichia coli, its thiolase enzyme activity, and the amino acid sequence homology search revealed that pBSGT-3 encodes an AT. The recombinant AT (r-thiolase) was expressed in an active form in an E. coli expression system, and purified to homogeneity by selective ammonium sulfate fractionation and two steps of column chromatography. The purified enzyme exhibited a specific activity of 4.70 mU/mg protein. Its N-terminal sequence was (NH₂)-Arg-Met-Tyr-Thr-Thr-Ala-Lys-Asn-Leu-Glu-, which corresponds to the sequence from positions 15 to 24 of the amino acid sequence deduced from pBSGT-3 clone. The r-thiolase in the inclusion body expressed highly in E. coli was the precursor form, which is slightly larger than the purified r-thiolase. When incubated with the cell-free extract of Dictyostelium cells, the precursor was converted to the same size to the purified r-thiolase, suggesting that the presequence at the N-terminus is removed by a Dictyostelium processing peptidase.     

Functional differences in yeast protein disulfide isomerases

PDI1 is the essential gene encoding protein disulfide isomerase in yeast. The Saccharomyces cerevisiae genome, however, contains four other nonessential genes with homology to PDI1: MPD1, MPD2, EUG1, and EPS1. We have investigated the effects of simultaneous deletions of these genes. In several cases, we found that the ability of the PDI1 homologues to restore viability to a pdi1-deleted strain when overexpressed was dependent on the presence of low endogenous levels of one or more of the other homologues. This shows that the homologues are not functionally interchangeable. In fact, Mpd1p was the only homologue capable of carrying out all the essential functions of Pdi1p. Furthermore, the presence of endogenous homologues with a CXXC motif in the thioredoxin-like domain is required for suppression of a pdi1 deletion by EUG1 (which contains two CXXS active site motifs). This underlines the essentiality of protein disulfide isomerase-catalyzed oxidation. Most mutant combinations show defects in carboxypeptidase Y folding as well as in glycan modification. There are, however, no significant effects on ER-associated protein degradation in the various protein disulfide isomerase-deleted strains.