Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Bactericidal/permeability-increasing protein (BPI) and BPI homologs at mucosal sites.

At mucosal surfaces, we must co-exist with a high density of diverse microorganisms; therefore, protection against these occurs on multiple levels. Leukocyte- and epithelial derived-antimicrobial peptides and proteins (AMPs) comprise an essential component of immune defense. These molecules possess antibacterial, antifungal and signalling properties and probably contribute to defence and maintenance of homeostasis between the host and commensal microorganisms. Among these AMPs is bactericidal/permeability-increasing protein (BPI), an antimicrobial protein with potent endotoxin-neutralising activity, and several homologs. This review explores the roles of BPI and and its homologs at the mucosal interface. Congeners of BPI are under biopharmaceutical development as novel anti-infective agents, highlighting the potential therapeutic relevance of this protein family.

Context-dependent dual role of SKI8 homologs in mRNA synthesis and turnover.

Eukaryotic mRNA transcription and turnover is controlled by an enzymatic machinery that includes RNA polymerase II and the 3' to 5' exosome. The activity of these protein complexes is modulated by additional factors, such as the nuclear RNA polymerase II-associated factor 1 (Paf1c) and the cytoplasmic Superkiller (SKI) complex, respectively. Their components are conserved across uni- as well as multi-cellular organisms, including yeast, Arabidopsis, and humans. Among them, SKI8 displays multiple facets on top of its cytoplasmic role in the SKI complex. For instance, nuclear yeast ScSKI8 has an additional function in meiotic recombination, whereas nuclear human hSKI8 (unlike ScSKI8) associates with Paf1c. The Arabidopsis SKI8 homolog VERNALIZATION INDEPENDENT 3 (VIP3) has been found in Paf1c as well; however, whether it also has a role in the SKI complex remains obscure so far. We found that transgenic VIP3-GFP, which complements a novel vip3 mutant allele, localizes to both nucleus and cytoplasm. Consistently, biochemical analyses suggest that VIP3-GFP associates with the SKI complex. A role of VIP3 in the turnover of nuclear encoded mRNAs is supported by random-primed RNA sequencing of wild-type and vip3 seedlings, which indicates mRNA stabilization in vip3. Another SKI subunit homolog mutant, ski2, displays a dwarf phenotype similar to vip3. However, unlike vip3, it displays neither early flowering nor flower development phenotypes, suggesting that the latter reflect VIP3's role in Paf1c. Surprisingly then, transgenic ScSKI8 rescued all aspects of the vip3 phenotype, suggesting that the dual role of SKI8 depends on species-specific cellular context.

Characterization of the mammalian homologs of the Drosophila Melanogaster Endocytic protein lethal (2) giant discs 1

Endocytose joue un rôle dans l'activation du récepteur Notch. Des mutations dans le gène drosophilien lethal giant discs (lgd), provoque une prolifération cellulaire en perturbant l'endocytose de Notch. Les orthologues murins mlgd1 et 2 peuvent sauver ce phénotype, démontrant une fonction conservée. Cependant, des publications récentes suggèrent que les orthologs humains de lgd (hgd1/2) sont nucléaires. Dans cette étude, il est démontré que chez la Drosophile, le mutant dlgd(08) provoque l'accumulation de Notch dans des vésicules et une surprolifération de neuroblastes . Ceci suggère que Notch est activé a l'intérieur des endosomes dans les neuroblastes. L'immunohistochimie de cellules Hela indique que hlgd1 et 2 ne sont pas nucléaires, mais associés à des strctures endosomales. Enfin, la baisse d'expression par shRNA des gènes murins mlgd1 et mlgd2 provoque une différenciation accélérée des cellules souches hématopoïétiques dans la lignée lymphopoïèse T et bloque la transition DN3 / CD4+CD8+, suggérant une suractivation de Notch.

Strukturelle und funktionelle Charakterisierung des Dnmt2-Homologs DnmA von Dictyostelium discoideum

In dieser Arbeit wurde die DNA Methyltransferase 2 aus Dictyostelium discoideum strukturell und funktionell untersucht. Sie vermittelt die Methylierung des Cytosin an Position 38 nahe des Anticodons der tRNAAsp (Goll et al., 2006; Müller et al., 2013). Jedoch ist die biologische Funktion dieser Methylierung bis heute nicht hinreichend geklärt. In der Vergangenheit konnten erhebliche Unterschiede in der in vivo- und in vitro-Methylierungsaktivität von DnmA, dem Dnmt2-Homolog aus D. discoideum, beobachtet werden. So wurde bis jetzt ausschließlich tRNAAsp als in vivo-Substrat des Proteins identifiziert. In vitro methyliert rekombinant gewonnenes DnmA aus E. coli allerdings auch Transkripte der tRNAGlu und tRNAGly (Müller et al., 2013). Aus diesem Grund sollten in dieser Arbeit posttranslationaler Proteinmodifikationen von DnmA identifiziert werden. Diese können an dem Protein aus D. discoideum, jedoch nicht oder verändert an dem Protein aus E. coli auftreten und deshalb zu einer abweichenden Methylierungsaktivität von DnmA führen. Es konnte gezeigt werden, dass DnmA aus D. discoideum isoliert, zahlreiche Proteinmodifikationen aufweist, wobei elf Methylierungen, drei Phosphorylierungen und drei Acetylierungen identifiziert werden konnten. Die als methyliert identifizierten Aminosäuren K205, K236, K276 und R341 und die phosphorylierte Aminosäure T239 wurden detaillierter untersucht. Dazu wurden sie jeweils zu Alanin mutiert. Keine der Aminosäureaustausch-mutationen führte zu einem erheblichen Strukturverlust des Proteins und alle Proteine zeigten in vitro-Methylierungsaktivität mit tRNAAsp, tRNAGlu und tRNAGly. Die Mutations-proteine DnmAK276A und R341A wurden überdies in vivo untersucht und zeigten eine verringerte Methylierungsaktivität. Diese Ergebnisse liefern erste Hinweise darauf, dass posttranslationale Proteinmodifikationen die Aktivität von DnmA in vivo beeinflussen könnten. Außerdem konnte gezeigt werden, dass auch DnmA, welches in D. discoideum überexprimiert und daraus gereinigt wurde, in vitro-Methylierungsaktivität mit tRNAGly besitzt. Da tRNAGly kein Substrat für DnmA in vivo darstellt (Müller et al., 2013), konnte dadurch nachgewiesen werden, dass das Protein, auch wenn es in D. discoideum exprimiert wird, in vivo und in vitro abweichende Substratspezifität aufweist. Weiterhin wurde versucht, Protein-Interaktionspartner von DnmA zu identifizieren. Mittels Immunpräzipitation und anschließender massenspektrometrischer Analyse konnten eine Vielzahl von Kandidaten identifiziert werden. Erste Versuche, die Ergebnisse zu verifizieren, zeigten allerdings keine Bestätigung der direkten Interaktion der Proteine CulB, CulE und Nola1 mit DnmA. Wie in vorherigen Untersuchungen (Dissertation Vladimir Maksimov, 2010; Dissertation Sara Müller, 2011), konnten auch im Rahmen dieser Arbeit keine direkt mit DnmA assoziierten Proteine in D. discoideum identifiziert werden. Im dritten Projekt der vorliegenden Arbeit wurde ein Zusammenhang zwischen der DnmA-vermittelten tRNA-Methylierung am C38 und einer weiteren tRNA-Modifikation, Queuosin an Position 34, gezeigt. Die Methylierung der tRNAAsp durch DnmA war signifikant erhöht, wenn das Nährmedium von D. discoideum mit Queuin supplementiert wurde. Allerdings ist Queuosin für die DnmA-vermittelte tRNA-Methylierung nicht unabdingbar. In vivo konnte nach Überexpression von DnmA ebenfalls eine Queuosin-unabhängige tRNAAsp-Methylierung detektiert werden. Weiterhin wurde gezeigt, dass Queuosin-enthaltende tRNAs kein generelles Substrat für DnmA darstellen. Nach Gabe von Queuin wiesen die anderen Queuosin-enthaltenden tRNAs tRNAAsn, tRNAHis und tRNATyr keine Methylierung putativer DnmA-targets auf. Auch an tRNAGlu und tRNAGly konnte unter diesen Bedingungen keine in vivo-Methylierung des C38 bzw. C37 gezeigt werden. Weiterhin wurden erste Untersuchungen zur Bestimmung der Funktion der DnmA-vermittelten Methylierung in Zusammenhang mit der Queuosin-Modifikation durchgeführt. Bereits 1985 wurde beschrieben, dass Queuin im Nährmedium die tRNA-Menge von tRNAAsp und tRNATyr in Wildtyp Dictyostelium-Zellen um das Zweifache erhöht (Ott and Kersten, 1985). Hier konnten diese Ergebnisse bestätigt und außerdem gezeigt werden, dass dieser Effekt in einem dnmA- -Stamm nicht auftritt. Interessanterweise stellte tRNATyr jedoch kein Methylierungssubstrat für DnmA dar. Dennoch konnte in einem dnmA- -Stamm ebenfalls keine erhöhte Menge dieser tRNA beobachtet werden, obwohl die Zellen mit Queuin kultiviert wurden. In wieweit ein indirekter Effekt, welcher nicht die DnmA-vermittelte Methylierung darstellt, weitere tRNA-Modifikationen oder andere Proteine an diesem Regulationsmechanismus beteiligt sind, muss in zukünftigen Analysen geklärt werden.

Type III secretion homologs are present in Brucella melitensis, B-ovis, and B-suis biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role.

Rhamnolipids Production by a Pseudomonas eruginosa LBI Mutant: Solutions and Homologs Characterization

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Properties and secretory mechanism of Musca domestica digestive chymotrypsin and its relation with Drosophila melanogaster homologs

Musca domestica larvae present two different digestive chymotryptic activities found in the posterior midgut (PMG): one major soluble activity in the lumen and another minor present in cell membrane fractions. Both soluble and membrane-bound chymotryptic activities have different half lives of thermal inactivation (46 degrees C) in the presence and absence of 10 mM Triton X-100, indicating that they are two different molecular species. Purified soluble chymotryptic activity has pH optimum 7.4 and a molecular mass of 28 kDa in SDS-PAGE. It does not cleave short substrates, such as Suc-F-MCA, preferring longer substrates, such as Suc-AAPF-MCA, with a primary specificity (kcat/Km) for Phe rather than Tyr and Leu residues. In-gel activity revealed a unique band against S-AAPF-MCA with the same migration as purified chymotrypsin. One chymotrypsinogen-like sequence (MdChy1) was sequenced, cloned and recombinantly expressed in Escherichia coli (DE3) Star. MdChy1 is expressed in the proximal posterior midgut (PMG1), as seen by RT-PCR. Expression analysis of other chymotrypsin genes revealed genes expressed at the anterior midgut (AMG) and PMG. Western blot of M. domestica midgut tissues using anti-MdChy1 antiserum showed a single band in samples from AMG and PMG, co-migrating with recombinant and purified enzymes. Immunogold labeling corresponding to Mdchy1 was found in small vesicles (thus indicating exocytosis) and in the lumen of AMG and PMG, corroborating the existence of two similar groups of chymotrypsins. Transcriptomes of M. domestica AMG and whole midgut prepared by pyrosequencing disclosed 41 unique sequences of chymotrypsin-like enzymes (19 probably functional), from which MdChy1 is highly expressed. Phylogenetic reconstruction of Drosophila melanogaster and M. domestica chymotrypsin-like sequences revealed that the chymotrypsin genes expanded before the evolutionary separation of Musca and Drosophila. (C) 2012 Elsevier Ltd. All rights reserved.

A conserved role for p48 homologs in protecting dopaminergic neurons from oxidative stress.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder characterized by the progressive loss of dopaminergic (DA) neurons. Both environmental and genetic factors are thought to contribute to the pathogenesis of PD. Although several genes linked to rare familial PD have been identified, endogenous risk factors for sporadic PD, which account for the majority of PD cases, remain largely unknown. Genome-wide association studies have identified many single nucleotide polymorphisms associated with sporadic PD in neurodevelopmental genes including the transcription factor p48/ptf1a. Here we investigate whether p48 plays a role in the survival of DA neurons in Drosophila melanogaster and Caenorhabditis elegans. We show that a Drosophila p48 homolog, 48-related-2 (Fer2), is expressed in and required for the development and survival of DA neurons in the protocerebral anterior medial (PAM) cluster. Loss of Fer2 expression in adulthood causes progressive PAM neuron degeneration in aging flies along with mitochondrial dysfunction and elevated reactive oxygen species (ROS) production, leading to the progressive locomotor deficits. The oxidative stress challenge upregulates Fer2 expression and exacerbates the PAM neuron degeneration in Fer2 loss-of-function mutants. hlh-13, the worm homolog of p48, is also expressed in DA neurons. Unlike the fly counterpart, hlh-13 loss-of-function does not impair development or survival of DA neurons under normal growth conditions. Yet, similar to Fer2, hlh-13 expression is upregulated upon an acute oxidative challenge and is required for the survival of DA neurons under oxidative stress in adult worms. Taken together, our results indicate that p48 homologs share a role in protecting DA neurons from oxidative stress and degeneration, and suggest that loss-of-function of p48 homologs in flies and worms provides novel tools to study gene-environmental interactions affecting DA neuron survival.

Isolation and characterization of mammalian homologs of the Drosophila gene glial cells missing

The glial cells missing (gcm) gene in Drosophila encodes a transcription factor that determines the choice between glial and neuronal fates. We report here the isolation of two mammalian gcm homologs, Gcm1 and Gcm2, and the characterization of their expression patterns during embryonic development. Although Gcm2 is expressed in neural tissues at a low level, the major sites of expression for both of the mammalian genes are nonneural, suggesting that the functions of the mammalian homologs have diverged and diversified. However, when expressed ectopically, Gcm1 can substitute functionally for Drosophila gcm by transforming presumptive neurons into glia. Thus, certain biochemical properties, although not the specificity of the tissue in which the gene is expressed, have been conserved through the evolution of the Gcm gene family.

A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: Diverse roles for mitogen-activated protein kinase homologs in foliar pathogens

Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development. Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important. We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways. The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae. A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia. No pseudothecia are formed when a cross between two Δchk1 mutants is attempted. The ability of Δchk1 mutants to infect corn plants is reduced severely. The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria. This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus. In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal. Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1. Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Signal transduction through homologs of the Ste20p and Ste7p protein kinases can trigger hyphal formation in the pathogenic fungus Candida albicans

The CST20 gene of Candida albicans was cloned by functional complementation of a deletion of the STE20 gene in Saccharomyces cerevisiae. CST20 encodes a homolog of the Ste20p/p65PAK family of protein kinases. Colonies of C. albicans cells deleted for CST20 revealed defects in the lateral formation of mycelia on synthetic solid “Spider” media. However, hyphal development was not impaired in some other media. A similar phenotype was caused by deletion of HST7, encoding a functional homolog of the S. cerevisiae Ste7p protein kinase. Overexpression of HST7 partially complemented the deletion of CST20. Cells deleted for CST20 were less virulent in a mouse model for systemic candidiasis. Our results suggest that more than one signaling pathway can trigger hyphal development in C. albicans, one of which has a protein kinase cascade that is analogous to the mating response pathway in S. cerevisiae and might have become adapted to the control of mycelial formation in asexual C. albicans.

Functional specificity of MutL homologs in yeast: Evidence for three Mlh1-based heterocomplexes with distinct roles during meiosis in recombination and mismatch correction

The yeast genome encodes four proteins (Pms1 and Mlh1–3) homologous to the bacterial mismatch repair component, MutL. Using two hybrid-interaction and coimmunoprecipitation studies, we show that these proteins can form only three types of complexes in vivo. Mlh1 is the common component of all three complexes, interacting with Pms1, Mlh2, and Mlh3, presumptively as heterodimers. The phenotypes of single deletion mutants reveal distinct functions for the three heterodimers during meiosis: in a pms1 mutant, frequent postmeiotic segregation indicates a defect in the correction of heteroduplex DNA, whereas the frequency of crossing-over is normal. Conversely, crossing-over in the mlh3 mutant is reduced to ≈70% of wild-type levels but correction of heteroduplex is normal. In a mlh2 mutant, crossing-over is normal and postmeiotic segregation is not observed but non-Mendelian segregation is elevated and altered with respect to parity. Finally, to a first approximation, the mlh1 mutant represents the combined single mutant phenotypes. Taken together, these data imply modulation of a basic Mlh1 function via combination with the three other MutL homologs and suggest specifically that Mlh1 combines with Mlh3 to promote meiotic crossing-over.