A multi-faceted approach to unravel coding and non-coding gene fusions and target chimeric proteins in ataxia.

Ataxia represents a heterogeneous group of neurodegenerative disorders characterized by a loss of balance and coordination, often resulting from mutations in genes vital for cerebellar function and maintenance. Recent advances in genomics have identified gene fusion events as critical contributors to various cancers and neurodegenerative diseases. However, their role in ataxia pathogenesis remains largely unexplored. Our study Hdelved into this possibility by analyzing RNA sequencing data from 1443 diverse samples, including cell and mouse models, patient samples, and healthy controls. We identified 7067 novel gene fusions, potentially pivotal in disease onset. These fusions, notably in-frame, could produce chimeric proteins, disrupt gene regulation, or introduce new functions. We observed conservation of specific amino acids at fusion breakpoints and identified potential aggregate formations in fusion proteins, known to contribute to ataxia. Through AI-based protein structure prediction, we identified topological changes in three high-confidence fusion proteins-TEN1-ACOX1, PEX14-NMNAT1, and ITPR1-GRID2-which could potentially alter their functions. Subsequent virtual drug screening identified several molecules and peptides with high-affinity binding to fusion sites. Molecular dynamics simulations confirmed the stability of these protein-ligand complexes at fusion breakpoints. Additionally, we explored the role of non-coding RNA fusions as miRNA sponges. One such fusion, RP11-547P4-FLJ33910, showed strong interaction with hsa-miR-504-5p, potentially acting as its sponge. This interaction correlated with the upregulation of hsa-miR-504-5p target genes, some previously linked to ataxia. In conclusion, our study unveils new aspects of gene fusions in ataxia, suggesting their significant role in pathogenesis and opening avenues for targeted therapeutic interventions.Communicated by Ramaswamy H. Sarma.

The Virulence Factor Macrophage Infectivity Potentiator (Mip) Influences Branched-Chain Amino Acid Metabolism and Pathogenicity of Legionella pneumophila.

Legionella pneumophila (Lp) is a common etiological agent of bacterial pneumonia that causes Legionnaires' disease (LD). The bacterial membrane-associated virulence factor macrophage infectivity potentiator (Mip) exhibits peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and contributes to the intra- and extracellular pathogenicity of Lp. Though Mip influences disease outcome, little is known about the metabolic consequences of altered Mip activity during infections. Here, we established a metabolic workflow and applied mass spectrometry approaches to decipher how Mip activity influences metabolism and pathogenicity. Impaired Mip activity in genetically engineered Lp strains decreases intracellular replication in cellular infection assays, confirming the contribution of Mip for Lp pathogenicity. We observed that genetic and chemical alteration of Mip using the PPIase inhibitors rapamycin and FK506 induces metabolic reprogramming in Lp, specifically branched-chain amino acid (BCAA) metabolism. Rapamycin also inhibits PPIase activity of mammalian FK506 binding proteins, and we observed that rapamycin induces a distinct metabolic signature in human macrophages compared to bacteria, suggesting potential involvement of Mip in normal bacteria and in infection. Our metabolic studies link Mip to alterations in BCAA metabolism and may help to decipher novel disease mechanisms associated with LD.

Protein sociology of ProA, Mip and other secreted virulence factors at the Legionella pneumophila surface.

The pathogenicity of L. pneumophila, the causative agent of Legionnaires' disease, depends on an arsenal of interacting proteins. Here we describe how surface-associated and secreted virulence factors of this pathogen interact with each other or target extra- and intracellular host proteins resulting in host cell manipulation and tissue colonization. Since progress of computational methods like AlphaFold, molecular dynamics simulation, and docking allows to predict, analyze and evaluate experimental proteomic and interactomic data, we describe how the combination of these approaches generated new insights into the multifaceted "protein sociology" of the zinc metalloprotease ProA and the peptidyl-prolyl cis/trans isomerase Mip (macrophage infectivity potentiator). Both virulence factors of L. pneumophila interact with numerous proteins including bacterial flagellin (FlaA) and host collagen, and play important roles in virulence regulation, host tissue degradation and immune evasion. The recent progress in protein-ligand analyses of virulence factors suggests that machine learning will also have a beneficial impact in early stages of drug discovery.

Legionella pneumophila PPIase Mip Interacts with the Bacterial Proteins SspB, Lpc2061, and FlaA and Promotes Flagellation.

The peptidyl-prolyl-cis/trans-isomerase (PPIase) macrophage infectivity potentiator (Mip) contributes to the pathogenicity and fitness of L. pneumophila, the causative agent of Legionnaires' disease. Here, we identified the stringent starvation protein SspB, hypothetical protein Lpc2061, and flagellin FlaA as bacterial interaction partners of Mip. The macrolide FK506, which inhibits the PPIase activity of Mip, interfered with the binding of Lpc2061. Moreover, we demonstrated that the N-terminal dimerization region and amino acid Y185 in the C-terminal PPIase domain of Mip are required for the binding of Lpc2061 and FlaA. The modeling of the interaction partners and global docking with Mip suggested nonoverlapping binding interfaces, and a molecular dynamic simulation predicted an increased stability for the tripartite interaction of Lpc2061, Mip, and FlaA. On the functional level, we demonstrated that Mip promotes L. pneumophila flagellation, which is positively influenced by the binding of Lpc2061 and reduced by FK506. Also, L. pneumophila mutants expressing the Y185A or the monomeric Mip variant, which bind less Lpc2061, were nonmotile, were less flagellated, and yielded less FlaA when quantified. To our knowledge, this is the first report in which a PPIase and its bacterial interaction partners were demonstrated to influence flagellation.

Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses.

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism.