Complexome Profiling—Exploring Mitochondrial Protein Complexes in Health and Disease

Complexome profiling (CP) is a state-of-the-art approach that combines separation of native proteins by electrophoresis, size exclusion chromatography or density gradient centrifugation with tandem mass spectrometry identification and quantification. Resulting data are computationally clustered to visualize the inventory, abundance and arrangement of multiprotein complexes in a biological sample. Since its formal introduction a decade ago, this method has been mostly applied to explore not only the composition and abundance of mitochondrial oxidative phosphorylation (OXPHOS) complexes in several species but also to identify novel protein interactors involved in their assembly, maintenance and functions. Besides, complexome profiling has been utilized to study the dynamics of OXPHOS complexes, as well as the impact of an increasing number of mutations leading to mitochondrial disorders or rearrangements of the whole mitochondrial complexome. Here, we summarize the major findings obtained by this approach; emphasize its advantages and current limitations; discuss multiple examples on how this tool could be applied to further investigate pathophysiological mechanisms and comment on the latest advances and opportunity areas to keep developing this methodology.

Expression of a phage-encoded Gp21 protein protects Pseudomonas aeruginosa against phage infection

There is a continuously expanding gap between predicted phage gene sequences and their corresponding functions, which largely hampered the development of phage therapy. Previous studies reported several phage proteins that could interfere with the intracellular processes of the host to obtain efficient infection. But few phage proteins that protect host against phage infection has been identified and characterized in detail. Here, we isolate a phage vB_Pae_QDWS capable of infecting Pseudomonas aeruginosa PAO1, and report its encoded Gp21 protein protects PAO1 against phage infection. Expressing of Gp21 regulate bacterial quorum sensing with an inhibitory effect in low cell density and activation effect in high cell density. By testing the TFPs-mediated twitching motility and transmission electron microscopy analysis, Gp21 was found decreased the pilus synthesis. Further constructing the TFPs synthesis gene pilB mutant and performing adsorption and phage resistance assay, we demonstrated Gp21 protein could block phage infection via decreasing the TFPs-mediated phage adsorption. Gp21 is a novel protein that inhibit phage efficacy against bacteria. The study deepens our understanding of phage-host interactions. Importance The majority of the annotated phage genes are currently deposited as “hypothetical protein” with unknown function. Researches revealed that some phage proteins serve to inhibit or redirect the host intracellular processes for phage infection. Differently, we report a phage encoded protein Gp21 that protect the host against phage infection. The pathways that Gp21 involved in anti-phage defense in Pseudomonas aeruginosa PAO1 are interfering with quorum sensing and decreasing the type IV pilus-mediated phage adsorption. Gp21 is a novel protein with a low sequence homology with other reported twitching inhibitory proteins. As a lytic phage derived protein, Gp21 expression protects P. aeruginosa PAO1 from reinfection by phage vB_Pae_QDWS, which may explain the well-known pseudolysogeny caused by virulent phages. Our discoveries provide valuable new insight into the phage-host evolutionary dynamics.

Predicting pathogenicity for novel hearing loss mutations based on genetic and protein structure approaches

Hearing loss is a heterogeneous disorder. Identification of causative mutations is demanding due to genetic heterogeneity. In this study, we investigated the genetic cause of sensorineural hearing loss in patients with severe/profound deafness. After the exclusion of GJB2-GJB6 mutations, we performed whole exome sequencing in 32 unrelated Argentinean families. Mutations were detected in 16 known deafness genes in 20 patients: ACTG1, ADGRV1 (GPR98), CDH23, COL4A3, COL4A5, DFNA5 (GSDDE), EYA4, LARS2, LOXHD1, MITF, MYO6, MYO7A, TECTA, TMPRSS3, USH2A and WSF1. Notably, 11 variants affecting 9 different non-GJB2 genes resulted novel: c.12829C > T, p.(Arg4277*) in ADGRV1; c.337del, p.(Asp109*) and c.3352del, p.(Gly1118Alafs*7) in CDH23; c.3500G > A, p.(Gly1167Glu) in COL4A3; c.1183C > T, p.(Pro395Ser) and c.1759C > T, p.(Pro587Ser) in COL4A5; c.580 + 2 T > C in EYA4; c.1481dup, p.(Leu495Profs*31) in LARS2; c.1939 T > C, p.(Phe647Leu), in MYO6; c.733C > T, p.(Gln245*) in MYO7A and c.242C > G, p.(Ser81*) in TMPRSS3 genes. To predict the effect of these variants, novel protein modeling and protein stability analysis were employed. These results highlight the value of whole exome sequencing to identify candidate variants, as well as bioinformatic strategies to infer their pathogenicity.

Inductive inference of novel protein-molecule interactions using Heterogeneous Graph Transformer (HGT) AutoEncoder

Protein-molecule interactions are promoted by the physicochemical characteristics of the actors involved, but structural information alone does not capture expression patterns, localization and pharmacokinetics. In this work we propose an integrative strategy for protein-molecule interaction discovery that combines different layers of information through the use of convolutional operators on graph, and frame the problem as missing link prediction task on an heterogeneous graph constituted by three node types: 1) molecules 2) proteins 3) diseases. Physicochemical information of the actors are encoded using shallow embedding techniques (SeqVec, Mol2Vec, Doc2Vec respectively) and are supplied as feature vectors to a Graph AutoEncoer (GAE) that uses a Heterogeneous Graph Transformer (HGT) in the encoder module. We show in this work that HGT Autoencoder can be used to accurately recapitulate the protein-molecule interactions set and propose novel relationships in inductive settings that are grounded in biological and functional information extracted from the graph.

Novel protein candidates for serodiagnosis of African animal trypanosomosis: Evaluation of the diagnostic potential of lysophospholipase and glycerol kinase from Trypanosoma brucei

African trypanosomosis, a parasitic disease caused by protozoan parasites transmitted by tsetse flies, affects both humans and animals in sub-Saharan Africa. While the human form (HAT) is now limited to foci, the animal form (AAT) is widespread and affects the majority of sub-Saharan African countries, and constitutes a real obstacle to the development of animal breeding. The control of AAT is hampered by a lack of standardized and easy-to used diagnosis tools. This study aimed to evaluate the diagnostic potential of TbLysoPLA and TbGK proteins from Trypanosoma brucei brucei for AAT serodiagnosis in indirect ELISA using experimental and field sera, individually, in combination, and associated with the BiP C-terminal domain (C25) from T. congolense. These novel proteins were characterized in silico, and their sequence analysis showed strong identities with their orthologs in other trypanosomes (more than 60% for TbLysoPLA and more than 82% for TbGK). TbLysoPLA displays a low homology with cattle (<35%) and Piroplasma (<15%). However, TbGK shares more than 58% with cattle and between 45–55% with Piroplasma. We could identify seven predicted epitopes on TbLysoPLA sequence and 14 potential epitopes on TbGK. Both proteins were recombinantly expressed in Escherichia coli. Their diagnostic potential was evaluated by ELISA with sera from cattle experimentally infected with T. congolense and with T.b. brucei, sera from cattle naturally infected with T. congolense, T. vivax and T.b. brucei. Both proteins used separately had poor diagnostic performance. However, used together with the BiP protein, they showed 60% of sensitivity and between 87–96% of specificity, comparable to reference ELISA tests. In conclusion, we showed that the performance of the protein combinations is much better than the proteins tested individually for the diagnosis of AAT.

A novel protein AXIN1-295aa encoded by circAXIN1 activates the Wnt/β-catenin signaling pathway to promote gastric cancer progression

Background Circular RNA (circRNA), a subclass of non-coding RNA, plays a critical role in cancer tumorigenesis and metastasis. It has been suggested that circRNA acts as a microRNA sponge or a scaffold to interact with protein complexes; however, its full range of functions remains elusive. Recently, some circRNAs have been found to have coding potential. Methods To investigate the role of circRNAs in gastric cancer (GC), parallel sequencing was performed using five paired GC samples. Differentially expressed circAXIN1 was proposed to encode a novel protein. FLAG-tagged circRNA overexpression plasmid construction, immunoblotting, mass spectrometry, and luciferase reporter analyses were applied to confirm the coding potential of circAXIN1. Gain- and loss-of-function studies were conducted to study the oncogenic role of circAXIN1 and AXIN1-295aa on the proliferation, migration, invasion, and metastasis of GC cells in vitro and in vivo. The competitive interaction between AXIN1-295aa and adenomatous polyposis coli (APC) was investigated by immunoprecipitation analyses. Wnt signaling activity was observed using a Top/Fopflash assay, real-time quantitative RT-PCR, immunoblotting, immunofluorescence staining, and chromatin immunoprecipitation. Results CircAXIN1 is highly expressed in GC tissues compared with its expression in paired adjacent normal gastric tissues. CircAXIN1 encodes a 295 amino acid (aa) novel protein, which was named AXIN1-295aa. CircAXIN1 overexpression enhances the cell proliferation, migration, and invasion of GC cells, while the knockdown of circAXIN1 inhibits the malignant behaviors of GC cells in vitro and in vivo. Mechanistically, AXIN1-295aa competitively interacts with APC, leading to dysfunction of the “destruction complex” of the Wnt pathway. Released β-catenin translocates to the nucleus and binds to the TCF consensus site on the promoter, inducing downstream gene expression. Conclusion CircAXIN1 encodes a novel protein, AXIN1-295aa. AXIN1-295aa functions as an oncogenic protein, activating the Wnt signaling pathway to promote GC tumorigenesis and progression, suggesting a potential therapeutic target for GC.