Biology subjects

Rogalski, J.

Publications and source records attributed to Rogalski, J..

7 recordsLinked to original sources

bioRxiv2026

Cathepsin K Mediates the Formation of Potential Rheumatoid Arthritis-Relevant Cis- and Trans-Spliced Peptides Compatible With HLA-DR4 Presentation

Rheumatoid arthritis (RA) is characterized by a loss of immunological tolerance to synovial self-proteins, yet the initial triggers generating novel neo-antigens remain incompletely defined. Here, we demonstrate that human cathepsin K (hCatK), a key cysteine protease driving joint degradation in RA, catalyzes covalent cis- and trans-splicing of peptides from major RA-associated self-proteins and foreign antigens, including type II collagen, fibrinogen, and SARS-CoV-2 Spike protein. Using high-resolution LC-MS/MS and database-assisted de novo sequencing, we identified over 90 unique spliced peptides. Splicing efficiency peaked at near-neutral pH (6.5-7.5), contrasting with classic hydrolytic profiles. Biochemical profiling revealed strong subsite selectivity, with a striking enrichment for small, aliphatic and/or hydroxyl-containing residues (Gly, Thr, Ser) at the P1 position. Furthermore, splicing preferentially targeted flexible, intrinsically disordered protein regions, with 81% of fibrinogen splicing events clustering within its C domain. In silico binding predictions for the RA-susceptibility allele HLA-DRB1*04:01 harboring the shared epitope revealed that numerous hCatK-generated spliced peptides exhibit predicted affinities exceeding those of established immunogenic and genomic sequences, uncovering protease-mediated transpeptidation as a novel post-translational modification capable of generating potent MHC class II autoantigens in RA.

biochemistry↗

bioRxiv2026

Recovering biological structure in sparse single-cell proteomics with GIRAFI

Single-cell proteomics (SCP) based on liquid-chromatography mass-spectrometry resolves protein-level cellular heterogeneity, but interpretation remains limited by detection-linked sparsity. SCP profiles continuous, peptide-derived intensities and has lower throughput than single-cell RNA sequencing, making denoising methods for large-scale, count-based transcriptomics difficult to apply. Here we present GIRAFI, a graph-informed statistical learning framework that imputes missing values and reveals reproducible cell states by constraining inference to dataset-aware, prior-knowledge-informed protein neighborhoods. We evaluated GIRAFI across SCP datasets spanning diverse biological/technical contexts. In masking-based recovery experiments and cell-type-specific protein-protein interaction inference, GIRAFI outperformed existing methods, and matched bulk proteomics comparisons corroborated recovery accuracy and ablations supported the graph-informed design. Beyond reduced replicate- and source-associated technical structure, GIRAFI recovered ground-truth cell-type annotations, improved cell state-resolved pathway analysis, and enabled trajectory inference consistent with known time courses. These results establish graph-constrained imputation as an effective strategy for improving SCP robustness, biological structure, interpretation, and cross-dataset comparability.

bioinformatics↗

bioRxiv2025

Beyond Degradation: How Reverse Proteolysis Creates Disease-Relevant Antigens

Proteases are conventionally regarded as degradative enzymes, yet their catalytic machinery also permits peptide bond formation through reverse proteolysis, a process that remains poorly characterized. Here, we show that lysosomal cysteine cathepsins catalyze iterative cycles of hydrolysis and ligation to generate multi-generational fusion peptides, including hybrids derived from host-viral protein substrates. Quantitative analysis demonstrates that peptide ligation can account for up to 4.5% of proteolytic turnover. This activity is strongly influenced by pH, substrate sequence, and post-translational modification, with citrullination and neutral pH favoring fusion peptide formation and the accumulation of more stable higher-order products. Using full-length protein substrates, we provide direct evidence that cathepsins can generate a hybrid insulin peptide previously identified as a Type 1 Diabetes (TID) autoantigen in patients. Moreover, several identified fusion peptides show effective binding to TID-associated HLA class II molecules. To examine whether ligation products can be captured under cellular conditions, we developed a click-based targeted transpeptide retrieval and purification strategy (CT-TRAP), which enabled detection of probe-derived cis/transpeptides in cell-based systems under controlled conditions. These findings establish reverse proteolysis by cysteine cathepsins as a quantifiable enzymatic pathway for generating non-genomically templated peptides, revealing an unrecognized dimension of lysosomal protease activity and peptide diversification.

biochemistry↗

bioRxiv2024

Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes

Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1. Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and 13C6 glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. 13C16 palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.

biochemistry↗

bioRxiv2024

Signal transduction pathways controlling Ins2 gene activity and β cell state transitions

Pancreatic {beta} cells exist in low and high insulin gene activity states that are dynamic on a scale of hours to days. Here, we used live 3D imaging, mass spectrometry proteomics, and targeted perturbations of {beta} cell signaling to comprehensively investigate Ins2(GFP)HIGH and Ins2(GFP)LOW {beta} cell states. We identified the two Ins2 gene activity states in intact isolated islets and showed that cells in the same state were more likely to be nearer to each other. We report the proteomes of pure {beta} cells to a depth of 5555 proteins and show that {beta} cells with high Ins2 gene activity had reduced {beta} cell immaturity factors, as well as increased translation. We identified activators of cAMP signaling (GLP1, IBMX) as powerful drivers of transitions from Ins2(GFP)LOW to the Ins2(GFP)HIGH states. Okadaic acid and cyclosporine A had the opposite effects. This study provides new insight into the proteomic profiles and regulation of {beta} cell states.

cell biology↗

bioRxiv2021

Insulin-dependent and -independent dynamics of insulin receptor trafficking in muscle cells

Insulin resistance contributes to type 2 diabetes and can be driven by hyperinsulinemia. Insulin receptor (INSR) internalization and cell-surface dynamics at rest and during insulin exposure are incompletely understood in muscle cells. Using surface labelling and live-cell imaging, we observed robust basal internalization of INSR in C2C12 myoblasts, without an effect of added insulin. Mass-spectrometry using INSR knockout cells as controls, identified high-confidence binding partners, including proteins associated with internalization. We confirmed known interactors, including IGF1R, but also identified underappreciated INSR-binding factors such as ANXA2. AlphaFold-Multimer analysis of these INSR-binding proteins predicted potential INSR binding sites of these proteins. Protein-protein interaction network mapping suggested links between INSR and caveolin-mediated endocytosis. INSR interacted with both caveolin and clathrin heavy chain (CLTC) in mouse skeletal muscle and C2C12 myoblasts. Whole cell 2D super-resolution imaging revealed that high levels of insulin (20 nM) increased INSR colocalization with CAV1 but decreased its colocalization with CLTC. Single particle tracking confirmed the colocalization of cell-surface INSR with both over-expressed CAV1-mRFP and CLTC-mRFP. INSR tracks that colocalized with CAV1 exhibited longer radii and lifetimes, regardless of insulin exposure, compared to non-colocalized tracks, whereas insulin further increased the lifetime of INSR/CLTC colocalized tracks. Overall, these data suggest that muscle cells utilize both CAV1 and CLTC-dependent pathways for INSR dynamics and internalization.

cell biology↗

bioRxiv2021

Proteomic portraits reveal evolutionarily conserved and divergent responses to spinal cord injury

Despite the emergence of promising therapeutic approaches in preclinical studies, the failure of large-scale clinical trials leaves clinicians without effective treatments for acute spinal cord injury (SCI). These trials are hindered by their reliance on detailed neurological examinations to establish outcomes, which inflate the time and resources required for completion. Moreover, therapeutic development takes place in animal models whose relevance to human injury remains unclear. Here, we address these challenges through targeted proteomic analyses of CSF and serum samples from 111 acute SCI patients and, in parallel, a large animal (porcine) model of SCI. We develop protein biomarkers of injury severity and recovery, including a prognostic model of neurological improvement at six months with an AUC of 0.91, and validate these in an independent cohort. Through cross-species proteomic analyses, we dissect evolutionarily conserved and divergent aspects of the SCI response, and establish the CSF abundance of glial fibrillary acidic protein (GFAP) as a biochemical outcome measure in both humans and pigs. Our work opens up new avenues to catalyze translation by facilitating the evaluation of novel SCI therapies, while also providing a resource from which to direct future preclinical efforts.

neuroscience↗