Unveiling the Peptidase Network Orchestrating Hemoglobin Catabolism in Rhodnius prolixus.

Chagas disease is transmitted to humans by obligatory hematophagous insects of Triatominae subfamily, which feeds on various hosts to acquire their nutritional sustenance derived from blood proteins. Hemoglobin (Hb) digestion is a pivotal metabolic feature of triatomines, representing a key juncture in their competence toward Trypanosoma cruzi; however, it remains poorly understood. To explore the Hb digestion pathway in Rhodnius prolixus, a major Chagas disease vector, we employed an array of approaches for activity profiling of various midgut-associated peptidases using specific substrates and inhibitors. Dissecting the individual contribution of each peptidase family in Hb digestion has unveiled a predominant role played by aspartic proteases and cathepsin B-like peptidases. Determination of peptidase-specific cleavage sites of these key hemoglobinases, in conjunction with mass spectrometry-based identification of in vivo Hb-derived fragments, has revealed the intricate network of peptidases involved in the Hb digestion pathway. This network is initiated by aspartic proteases and subsequently sustained by cysteine proteases belonging to the C1 family. The process is continued simultaneously by amino and carboxypeptidases. The comprehensive profiling of midgut-associated aspartic proteases by quantitative proteomics has enabled the accurate revision of gene annotations within the A1 family of the R. prolixus genome. Significantly, this study also serves to illuminate a potentially important role of the anterior midgut in blood digestion. The expanded repertoire of midgut-associated proteases presented in this study holds promise for the identification of novel targets aimed at controlling the transmission of Chagas disease.

Disclosing the Interactome of Leukemogenic NUP98-HOXA9 and SET-NUP214 Fusion Proteins Using a Proteomic Approach.

The interaction of oncogenes with cellular proteins is a major determinant of cellular transformation. The NUP98-HOXA9 and SET-NUP214 chimeras result from recurrent chromosomal translocations in acute leukemia. Functionally, the two fusion proteins inhibit nuclear export and interact with epigenetic regulators. The full interactome of NUP98-HOXA9 and SET-NUP214 is currently unknown. We used proximity-dependent biotin identification (BioID) to study the landscape of the NUP98-HOXA9 and SET-NUP214 environments. Our results suggest that both fusion proteins interact with major regulators of RNA processing, with translation-associated proteins, and that both chimeras perturb the transcriptional program of the tumor suppressor p53. Other cellular processes appear to be distinctively affected by the particular fusion protein. NUP98-HOXA9 likely perturbs Wnt, MAPK, and estrogen receptor (ER) signaling pathways, as well as the cytoskeleton, the latter likely due to its interaction with the nuclear export receptor CRM1. Conversely, mitochondrial proteins and metabolic regulators are significantly overrepresented in the SET-NUP214 proximal interactome. Our study provides new clues on the mechanistic actions of nucleoporin fusion proteins and might be of particular relevance in the search for new druggable targets for the treatment of nucleoporin-related leukemia.

Long-term antigen exposure irreversibly modifies metabolic requirements for T cell function.

Energy metabolism is essential for T cell function. However, how persistent antigenic stimulation affects T cell metabolism is unknown. Here, we report that long-term in vivo antigenic exposure induced a specific deficit in numerous metabolic enzymes. Accordingly, T cells exhibited low basal glycolytic flux and limited respiratory capacity. Strikingly, blockade of inhibitory receptor PD-1 stimulated the production of IFNγ in chronic T cells, but failed to shift their metabolism towards aerobic glycolysis, as observed in effector T cells. Instead, chronic T cells appeared to rely on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO) to produce ATP for IFNγ synthesis. Check-point blockade, however, increased mitochondrial production of superoxide and reduced viability and effector function. Thus, in the absence of a glycolytic switch, PD-1-mediated inhibition appears essential for limiting oxidative metabolism linked to effector function in chronic T cells, thereby promoting survival and functional fitness.

Comparative proteome profiling of hydatid fluid from Algerian patients reveals cyst location-related variation in Echinococcus granulosus.

Human cystic echinococcosis, an endemic zoonosis in Algeria, is caused by larvae of the cestode Echinococcus granulosus. Parasitic modulation of the immune response allows E. granulosus to persist in intermediate hosts. Previous in vitro and in vivo immunological studies have shown differences in host immune responses according to the status and location of the hydatid cysts in the body. In this study, a proteomic analysis of human hydatid fluids was performed to identify the proteins in hydatid cyst fluids. Hydatid fluid was obtained after cystic surgical removal from three patients with these cysts. The study was conducted on fertile hydatid fluids from lungs, vertebra, and infertile paravertebral fluids. Comparisons of the protein compositions of these fluids revealed differences in their protein profiles. These differences are probably related to the cyst location and fertility status of the parasite. Notably, our analysis identified new proteins from the parasite and human host. The identification of host proteins in hydatid fluids indicates that the hydatid walls are permeable allowing a high protein exchange rate between the metacestode and the affected tissue. Interestingly, our study also revealed that parasite antigenic protein expression variations reflect the differences observed in host immunostimulation.

An insight into the transcriptome of the digestive tract of the bloodsucking bug, Rhodnius prolixus.

The bloodsucking hemipteran Rhodnius prolixus is a vector of Chagas' disease, which affects 7-8 million people today in Latin America. In contrast to other hematophagous insects, the triatomine gut is compartmentalized into three segments that perform different functions during blood digestion. Here we report analysis of transcriptomes for each of the segments using pyrosequencing technology. Comparison of transcript frequency in digestive libraries with a whole-body library was used to evaluate expression levels. All classes of digestive enzymes were highly expressed, with a predominance of cysteine and aspartic proteinases, the latter showing a significant expansion through gene duplication. Although no protein digestion is known to occur in the anterior midgut (AM), protease transcripts were found, suggesting secretion as pro-enzymes, being possibly activated in the posterior midgut (PM). As expected, genes related to cytoskeleton, protein synthesis apparatus, protein traffic, and secretion were abundantly transcribed. Despite the absence of a chitinous peritrophic membrane in hemipterans - which have instead a lipidic perimicrovillar membrane lining over midgut epithelia - several gut-specific peritrophin transcripts were found, suggesting that these proteins perform functions other than being a structural component of the peritrophic membrane. Among immunity-related transcripts, while lysozymes and lectins were the most highly expressed, several genes belonging to the Toll pathway - found at low levels in the gut of most insects - were identified, contrasting with a low abundance of transcripts from IMD and STAT pathways. Analysis of transcripts related to lipid metabolism indicates that lipids play multiple roles, being a major energy source, a substrate for perimicrovillar membrane formation, and a source for hydrocarbons possibly to produce the wax layer of the hindgut. Transcripts related to amino acid metabolism showed an unanticipated priority for degradation of tyrosine, phenylalanine, and tryptophan. Analysis of transcripts related to signaling pathways suggested a role for MAP kinases, GTPases, and LKBP1/AMP kinases related to control of cell shape and polarity, possibly in connection with regulation of cell survival, response of pathogens and nutrients. Together, our findings present a new view of the triatomine digestive apparatus and will help us understand trypanosome interaction and allow insights into hemipteran metabolic adaptations to a blood-based diet.