A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

Six-week anaerobic training improves proteolytic profile of diabetic rats

Objective To evaluate the effect of six-week anaerobic training on the mRNA expression of genes related to proteolysis Ubb (Ubiquitin), E2-14kDa, Trim63 (MuRF1 protein) and Nfkb1 in the skeletal muscle of diabetic rats.Materials and methods Four groups were established: DE (DiabetesExercised), DS (Diabetes Sedentary), CE (Control Exercised) and CS (Control Sedentary). The training consisted of 3 sets of 12 jumps in the liquid mean with load equivalent to 50% of BW for 6 weeks. Euthanasia occurred under ip anesthesia, and blood, adipose tissue and skeletal muscles were collected. Gene expression was quantified by RT–PCR in the gastrocnemius muscle. ANOVA one-way was used for comparison among groups, with post-hoc (Tukey) when necessary, considering p < 0.05.Results We observed reduction in the body weight and adipose tissue in the diabetic groups. The muscle mass was reduced in DS, which could be reversed by training (DE). Although DS and DE have presented similar body weight, the training protocol in DE promoted reduction in the adipose tissue, and increase of muscle mass. Anaerobic training was efficient to reduce glycaemia only in the diabetic animals until 6 hours after the end of training. The Trim63 gene expression was increased in DS; decreased Ubb gene level was observed in trained rats (CE and DE) compared to sedentary (CS and DS), and DE presented the lowest level of E2-14kDa gene expression.Conclusion Six-week anaerobic training promoted muscle mass gain, improved glycemic control, and exerted inhibitory effect on the proteolysis of gastrocnemius muscle of diabetic rats.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime(R)) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in a resistant isolate compared to a sensitive one. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.