NEK5 interacts with LonP1 and its kinase activity is essential for the regulation of mitochondrial functions and mtDNA maintenance.

Little is known about Nima-related kinase (NEKs), a widely conserved family of kinases that have key roles in cell-cycle progression. Nevertheless, it is now clear that multiple NEK family members act in networks, not only to regulate specific events of mitosis, but also to regulate metabolic events independently of the cell cycle. NEK5 was shown to act in centrosome disjunction, caspase-3 regulation, myogenesis, and mitochondrial respiration. Here, we demonstrate that NEK5 interacts with LonP1, an AAA+ mitochondrial protease implicated in protein quality control and mtDNA remodeling, within the mitochondria and it might be involved in the LonP1-TFAM signaling module. Moreover, we demonstrate that NEK5 kinase activity is required for maintaining mitochondrial mass and functionality and mtDNA integrity after oxidative damage. Taken together, these results show a new role of NEK5 in the regulation of mitochondrial homeostasis and mtDNA maintenance, possibly due to its interaction with key mitochondrial proteins, such as LonP1.

Expression profile and subcellular localization of HslV, the proteasome related protease from Trypanosoma cruzi.

Trypanosoma cruzi is a rare example of an eukaryote that has genes for two threonine proteases: HslVU complex and 20S proteasome. HslVU is an ATP-dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. In this study, we expressed and obtained specific antibodies to HslU and HslV recombinant proteins and demonstrated the interaction between HslU/HslV by coimmunoprecipitation. To evaluate the intracellular distribution of HslV in T. cruzi we used an immunofluorescence assay and ultrastructural localization by transmission electron microscopy. Both techniques demonstrated that HslV was localized in the kinetoplast of epimastigotes. We also analyzed the HslV/20S proteasome co-expression in Y, Berenice 62 (Be-62) and Berenice 78 (Be-78) T. cruzi strains. Our results showed that HslV and 20S proteasome are differently expressed in these strains. To investigate whether a proteasome inhibitor could modulate HslV and proteasome expressions, epimastigotes from T. cruzi were grown in the presence of PSI, a classical proteasome inhibitor. This result showed that while the level of expression of HslV/20S proteasome is not affected in Be-78 strain, in Y and Be-62 strains the presence of PSI induced a significantly increase in Hslv/20S proteasome expression. Together, these results suggest the coexistence of the protease HslVU and 20S proteasome in T. cruzi, reinforcing the hypothesis that non-lysosomal degradation pathways have an important role in T. cruzi biology.

FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex.

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino-terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two-step processing mechanism that resulted in an amino-proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl-truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide-swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.

Selección de cepas nativas con actividad quitino-proteolítica de bacillus sp aisladas de suelos tropicales / Selecting native Bacillus sp strains having chitinolytic and proteolytic activity which have been isolated from tropical soils

El objetivo de este trabajo fue la selección de cepas nativas del género Bacillus con actividad quitinolíticay proteolítica, en suelo tropical en la costa de Oaxaca, México. Se aislaron 150 cepas, de las cuales 22fueron seleccionadas por presentar actividad quitinolítica y proteolítica. Dicha actividad se evaluó porla formación de halo de hidrólisis alrededor de la colonia en medios de cultivo suplementados con quitinacoloidal al 5% y leche descremada al 1% respectivamente. Las cepas LUM B001, B003, B013, B015y B065 presentaron mayor actividad quitinolítica y proteolítica, por lo que tienen el potencial para serevaluadas en control biológico de hongos fitopatógenos. Se encontró al género Bacillus distribuido ensuelos cultivados y no cultivados, no se encontraron diferencias estadísticas según el cultivo establecido(P<0,05), sin embargo se encontraron diferencias significativas (P<0,05) entre las zonas estudiadas, correspondiendolas menores recuperaciones de cepas a los terrenos del municipio de Tututepec, Oaxaca.

This work was aimed at selecting native strains from the Bacillus genus having chitinolytic and proteolytic activityfrom soil from the tropical coast of Oaxaca, Mexico. 150 strains were isolated, 22 of which were selectedas they presented chitinolytic and proteolytic activity. Such activity was assessed by the formation of a hydrolysishalo around the colony in culture media supplemented with 5% colloidal chitin and 1% skimmed milk.The LUM B001, B003, B013, B015 and B065-chitin strains presented higher quitinolytic and proteolytic activity,thereby having the potential for being evaluated in the biological control of phytopathogenic fungi. TheBacillus genus was found in cultivated and uncultivated soils; no statistical differences were found accordingto established crop (p <0.05); however, significant differences (p <0.05) were found between the areas beingstudied regarding the smaller amount of strains collected from land in the municipality of Tututepec, Oaxaca.

The Mycobacterium leprae hsp65 displays proteolytic activity. Mutagenesis studies indicate that the M. leprae hsp65 proteolytic activity is catalytically related to the HslVU protease.

The present study reports, for the first time, that the recombinant hsp65 from Mycobacterium leprae (chaperonin 2) displays a proteolytic activity toward oligopeptides. The M. leprae hsp65 proteolytic activity revealed a trypsin-like specificity toward quenched fluorescence peptides derived from dynorphins. When other peptide substrates were used (beta-endorphin, neurotensin, and angiotensin I), the predominant peptide bond cleavages also involved basic amino acids in P(1), although, to a minor extent, the hydrolysis involving hydrophobic and neutral amino acids (G and F) was also observed. The amino acid sequence alignment of the M. leprae hsp65 with Escherichia coli HslVU protease suggested two putative threonine catalytic groups, one in the N-domain (T(136), K(168), and Y(264)) and the other in the C-domain (T(375), K(409), and S(502)). Mutagenesis studies showed that the replacement of K(409) by A caused a complete loss of the proteolytic activity, whereas the mutation of K(168) to A resulted in a 25% loss. These results strongly suggest that the amino acid residues T(375), K(409), and S(502) at the C-domain form the catalytic group that carries out the main proteolytic activity of the M. leprae hsp65. The possible pathophysiological implications of the proteolytic activity of the M. leprae hsp65 are now under investigation in our laboratory.

Cloning and characterization of a LON gene homologue from the human pathogen Paracoccidioides brasiliensis.

A LON gene homologue from the human pathogen Paracoccidioides brasiliensis (PbLON) has been cloned, sequenced and characterized. It encodes a putative ATP-dependent proteinase Lon, which in Saccharomyces cerevisisae (PIM1) is a heat-inducible protein involved in the degradation of abnormal or short-lived proteins in the mitochondria. The PbLON ORF is within a 3369 bp fragment interrupted by two introns located in the 3'segment. The 5' and 3' regions flanking the ORF contain sequences which resemble known transcription elements. Several transcription binding factor motifs have also been found, including sites for heat shock/stress response and nitrogen control. The deduced protein consists of 1063 residues containing a mitochondrial import signal at the N-terminus and conserved ATP-binding (GPPGVGKT) and serine catalytic (KDGPSAG) sites. It shares high identity with Lon homologues from S. cerevisiae (73%), Homo sapiens (62%) and Escherichia coli (56%). In P. brasiliensis, an MDJ1 putative gene has also been partially sequenced adjacent to PbLON, possibly sharing divergently orientated promoter elements. This chromosomal organization is interesting, since Mdj1p is a heat shock chaperone essential for substrate degradation by PIM1 in yeast.

The Amazonia variant of Vibrio cholerae: molecular identification and study of virulence genes.

The pathogenic O1 Amazonia variant of Vibrio cholerae has been shown previously to have a cytotoxin acting on cultured Vero and Y-1 cells, and to lack important virulence factors such as the cholera toxin (Coelho et al. 1995a). This study extends the molecular analysis of the Amazonia strains, detecting the presence of the toxR gene, with a very similar sequence to that of the E1 Tor and classical biotypes. The outer membrane proteins are analyzed, detecting a variation among the group of Amazonia strains, with three different patterns found. As a by-product of this work a polymerase chain reaction fragment was sequenced, reading part of the sequence of the Lon protease of the Amazonia strains. This gene was not previously described in V. cholerae, but its sequence is present in the TIGR database specific for this species.

Cloning, nucleotide sequencing, and expression of the Azospirillum brasilense lon gene: involvement in iron uptake.

The lon gene of Escherichia coli encodes the lon (La) protease, which is associated with cellular protein degradation. A lon gene homolog from Azospirillum brasilense, a nitrogen-fixing soil bacterium which lives in association with the roots of cereal grasses, was cloned and characterized. The nucleotide sequence of the A. brasilense lon gene was determined. It contains an open reading frame that encodes a protein of 810 amino acids with a predicted molecular mass of about 90 kDa. The deduced amino acid sequence showed a high level of homology with the sequences of all the known lon gene products. An open reading frame homologous to the E. coli clpX gene was found in front of the lon gene. Transcriptional analysis showed that the lon gene of A. brasilense is induced by heat shock and that the mRNA is monocistronic. An A. brasilense mutant, with Tn5 inserted in the lon gene, was shown to be defective in iron uptake and failed to express two membrane proteins that are induced by iron starvation in the parental strain.

[Restoration of transcription antitermination of N- bacteriophages in Escherichia coli with a mutant RNA polymerase]. / Restauración de la antiterminación de la transcripción de bacteriófagos N-en Escherichia coli con una RNA polimerasa mutante.

The N protein of bacteriophage lambda modifies Escherichia coli RNA polymerase in such a way that it transcribes through termination signals, in a process called antitermination. In general N-mutants are not able to perform transcription antitermination. In this paper we report the suppression of N7 and Nmar3 mutations by Escherichia coli ron-lon strain. The lon mutation causes the N protein half-life to raise, suggesting that excess of N7 fragment or Nmar3 protein overcome the defect in antitermination. Under these conditions the lambda N-phages produced a titer similar to lambda wild type, although the plaques were smaller. These observations highlight the relevance of N half life in the regulation of transcription antitermination.