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TDP-43 pathology is associated with divergent protein profiles in ALS brain and spinal cord
Abstract Neuronal and glial cytoplasmic inclusions positive for TAR DNA-binding protein 43 (TDP-43) are the defining pathological hallmark of 97% of amyotrophic lateral sclerosis (ALS) and 50% of frontotemporal dementia (FTD). The ALS-FTD clinicopathological spectrum variably involves cortical and spinal anterior horn cell pathology. The broader protein composition of these inclusions is of major importance to understanding pathogenesis, clinical heterogeneity and biomarker development. This study examined the proteome associated with TDP-43 inclusions in ALS, using mass spectrometry-based proteomic analysis of spinal cord and cerebral cortex from donors with phosphoTDP-43 positive ALS (n = 16), alpha-synuclein positive Parkinson’s disease (PD, n = 8), phosphotau and beta-amyloid positive Alzheimer’s disease (AD, n = 8) and age matched non-neurological controls (n = 8), comparing ALS with non-ALS conditions, spinal cord with cerebral cortex samples, and detergent-soluble with -insoluble fractions. Increased abundance of TDP-43 in the detergent-insoluble fraction of ALS cortex and spinal cord tissue confirmed disease-specific protein enrichment by serial fractionation. The most striking alterations between ALS and other conditions were found in the detergent-insoluble fraction of spinal cord, with predominant enrichment of endosomal and extracellular vesicle pathways. In the cortex mitochondrial membrane/envelope and ion transmembrane transport pathways were enriched in the detergent-insoluble fraction. RNA/DNA metabolic processes (in spinal cord) versus mitochondrial and synaptic protein pathways (in cortex) were upregulated in the detergent-soluble fraction of ALS cases and downregulated in the insoluble protein fraction. Whilst motor cortex and spinal cord may not optimally reflect disease-specific pathways in AD, in PD a significant enrichment of alpha-synuclein in the detergent-insoluble fraction of spinal cord was found. Among proteins concordantly elevated in the detergent-insoluble fractions of spinal cord and cortex, there was greater representation of proteins encoded by ALS-associated genes, specifically Cu/Zn superoxide dismutase 1, valosin containing protein and TDP-43 (odds ratio 16.34, p = 0.002). No significant increase in TDP-43 interacting proteins was observed in either detergent-soluble or -insoluble fractions. Together, this study shows a divergence in the composition of proteins associated with TDP-43 positive detergent-insoluble inclusions between spinal cord and cerebral cortex. A common upregulation of proteins encoded by ALS-causing genes implicates their role in the pathogenesis of the ALS-FTD spectrum of diseases beyond TDP-43. Data are available via ProteomeXchange with identifier PXD067060.
Nitric oxide promotes cysteine N-degron proteolysis through control of oxygen availability
Selected proteins containing an N-terminal cysteine (Nt-Cys) are subjected to rapid, O 2 -dependent proteolysis via the Cys/Arg-branch of the N-degron pathway. Cysteine dioxygenation is catalyzed in mammalian cells by 2-aminoethanethiol dioxygenase (ADO), an enzyme that manifests extreme O 2 sensitivity. The canonical substrates of this pathway in mammalia are the regulators of G-protein signaling 4, 5, and 16, as well as interleukin-32. In addition to operating as an O 2 -sensing mechanism, this pathway has previously been described as a sensor of nitric oxide (NO), with robust effects on substrate stability upon modulation of NO bioavailability being widely demonstrated. Despite this, no mechanism to describe the action of NO on the Cys/Arg N-degron pathway has yet been substantiated. We demonstrate that NO can regulate the stability of Cys N-degron substrates indirectly via the regulation of ADO cosubstrate availability. Through competitive, O 2 -dependent inhibition of cytochrome C oxidase, NO can substantially modify cellular O 2 consumption rate and, in doing so, alter the availability of O 2 for Nt-Cys dioxygenation. We show that this increase in O 2 availability in response to NO exposure is sufficient to alter both dynamic and steady-state ADO substrate levels. It is likely that this mechanism operates to couple O 2 supply and mitochondrial respiration with responses to G-protein-coupled receptor stimulation.
A systems-based approach to uterine fibroids identifies differential splicing associated with abnormal uterine bleeding.
BackgroundUterine fibroids (UFs), benign tumours prevalent in up to 80% of women of reproductive age, are associated with significant morbidity, including abnormal uterine bleeding, pain and infertility. Despite identification of key genomic alterations in MED12 and HMGA2, the pathogenic mechanisms underlying UFs and heavy menstrual bleeding (HMB) remain poorly understood.MethodsTo correlate systematically genetic, transcriptional and proteomic phenotypes, we conducted an integrative multi-omic approach utilising targeted DNA sequencing, RNA sequencing and proteomic methodologies, encompassing fibroid, myometrium, and endometrium tissues from 91 patients.ResultsIn addition to confirming the presence of MED12 mutations, we identify variants in AHR and COL4A6. Multi-omic analysis of endometrium identifies latent factors that correlate with HMB and fibroid presence with driver mutations of MED12, AHR, and COL4A6, which are associated with pathways involved in angiogenesis, extracellular matrix organisation and RNA splicing. We propose a model, supported by in vivo evidence, where altered signalling of MED12-mutated fibroids influences RNA transcript isoform expression in endometrium, potentially leading to abnormal uterine bleeding.ConclusionsThis study presents a comprehensive integrative approach, revealing that genetic alterations in UF may influence endometrial function via signalling impacts on the RNA splicing mechanism. Our findings advance the understanding of complex molecular pathways in UF pathogenesis and UF-associated endometrial dysfunction, offering insights for targeted therapeutic development.
Pleural fluid proteomics from patients with pleural infection shows signatures of diverse neutrophilic responses: The Oxford Pleural Infection Endotyping Study (TORPIDS-2)
Background Pleural infection is a complex disease with poor clinical outcomes and increasing incidence worldwide, yet its biological endotypes remain unknown. Methods We analysed 80 pleural fluid samples from the PILOT study, a prospective study on pleural infection, using unlabelled mass spectrometry. A total of 449 proteins were retained after filtering. Unsupervised hierarchical clustering and Uniform Manifold Approximation and Projection analyses were used to cluster samples and pathway analysis was performed to identify the biological processes. Protein signatures as identified by the pathway analysis were compared to microbiology as defined by 16S rRNA next-generation sequencing. Spearman and exact Fischer's methods were used for correlation assessment. Results Higher neutrophil degranulation was correlated with increased glycolysis (odds ratio (OR) 281, p<2.2×10−16) and pentose phosphate activation (OR 371.45, p<2.2×10−16). Samples dominated by Streptococcus pneumoniae exhibited higher neutrophil degranulation (OR 12.08, p=0.005), glycolysis (OR 11.4, p=0.006) and pentose phosphate activity (OR 12.82, p=0.004). Samples dominated by anaerobes and Gram-negative bacteria exhibited lower neutrophil degranulation (OR 0.15, p=0.01), glycolysis (OR 0.14, p=0.01) and pentose phosphate activity (OR 0.07, p=0.001). Increased activity of the liver and retinoid X receptors pathway was associated with lower risk of 1-year mortality (OR 0.24, p=0.04). Conclusions These findings suggest that pleural infection patients exhibit diverse responses of neutrophil-mediated immunity, glycolysis and pentose phosphate activation, which are associated with microbiology. Therapeutic targeting of the liver and retinoid X receptors pathway with agonists is a possible treatment approach.
A new paradigm of islet adaptations in human pregnancy: insights from immunohistochemistry and proteomics
Abstract Physiological changes during pregnancy support foetal growth, including adaptations in pancreatic islets to maintain glucose homeostasis. We investigate these adaptations using rare, high-quality pancreatic tissue from pregnant human donors and matched controls. We profile islets from pregnant donors using proteomics and assess α- and β-cell characteristics, as well as prolactin receptor and serotonin 2B receptor expression. Proteomic profiling of microdissected human islets identifies 7546 proteins but shows minimal differences in protein expression. In pregnancy, we show that islet area increases 1.9-fold, α- and β-cell areas increase 4.3- and 1.9-fold, driven by an increase in cell number rather than hypertrophy. Prolactin receptor expression is higher in α but not β cells, and serotonin 2B receptor is undetectable in β cells. Glucagon-like peptide-1 abundance increases 2.9-fold in α cells. These findings indicate that the molecular mechanisms driving pregnancy-induced islet adaptations in humans differ from those in mice, highlighting the need for human-based studies.
Amyloid-β disrupts APP-regulated protein aggregation and dissociation from recycling endosomal membranes.
Secretory proteins aggregate into non-soluble dense-core granules in recycling endosome-like compartments prior to regulated release. By contrast, aberrantly processed, secreted amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) form pathological extracellular amyloidogenic aggregations in late-stage Alzheimer's disease (AD). By examining living Drosophila prostate-like secondary cells, we show that both APP and Aβ peptides affect normal biogenesis of dense-core granules. These cells generate dense-core granules and secreted nanovesicles called Rab11-exosomes via evolutionarily conserved mechanisms within highly enlarged secretory compartments with recycling endosomal identity. The fly APP homologue, APP-like (APPL), associates with these vesicles and the compartmental limiting membrane, from where its extracellular domain modulates protein aggregation. Proteolytic release of this domain permits mini-aggregates to coalesce into a large central dense-core granule. Mutant Aβ expression disrupts this process and compartment motility, and increases aberrant lysosomal targeting, mirroring previously unexplained early-stage pathological events in AD. It also promotes cell-to-cell propagation of these endolysosomal defects, again phenocopying changes observed in AD. Our data therefore demonstrate physiological roles for APP in membrane-dependent protein aggregation, involving molecular mechanisms, which when disrupted by Aβ peptides, trigger Alzheimer's disease-relevant pathologies.
The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity
Background: Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work studies protein tyrosine kinases as well-known targets for successful cancer therapies. It focuses on Src family kinases (SFK), which are known to play a critical role in some head and neck tumors. Methods: Western blot analyses of phospho-tyrosine protein patterns in 34 TSCC lines facilitated the investigation of SFK as contributors to these phosphorylations. The SFK inhibitors PP2 and Dasatinib were utilized to determine SFK contributions to cell motility and survival. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses of synergistic effects of Dasatinib and glucocorticoids on TSCC lines. Effects on the cell cycle were investigated by flow cytometry and expression levels of cell cycle regulators. Senescence was analyzed by senescence-associated β galactosidase detection and p27Kip1 protein expression. Autophagy was measured by Acridine Orange staining. Results: A panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, appeared to increase senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins. Conclusion: This work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments.
Acidosis attenuates the hypoxic stabilization of HIF-1α by activating lysosomal degradation
Hypoxia-inducible factors (HIFs) mediate cellular responses to low oxygen, notably enhanced fermentation that acidifies poorly perfused tissues and may eventually become more damaging than adaptive. How pH feeds back on hypoxic signaling is unclear but critical to investigate because acidosis and hypoxia are mechanistically coupled in diffusion-limited settings, such as tumors. Here, we examined the pH sensitivity of hypoxic signaling in colorectal cancer cells that can survive acidosis. HIF-1α stabilization under acidotic hypoxia was transient, declining over 48 h. Proteomic analyses identified responses that followed HIF-1α, including canonical HIF targets (e.g., CA9, PDK1), but these did not reflect a proteome-wide downregulation. Enrichment analyses suggested a role for lysosomal degradation. Indeed, HIF-1α destabilization was blocked by inactivating lysosomes, but not proteasome inhibitors. Acidotic hypoxia stimulated lysosomal activity and autophagy via mammalian target of rapamycin complex I (mTORC1), resulting in HIF-1α degradation. This response protects cells from excessive acidification by unchecked fermentation. Thus, alkaline conditions are permissive for at least some aspects of HIF-1α signaling.
Activity-Based Protein Profiling (ABPP) of Cellular DeISGylating Enzymes and Inhibitor Screening.
A detailed methodology platform is described for activity-based protein profiling (ABPP) of cellular deISGylating enzymes using a specific activity-based interferon-stimulated gene 15 (ISG15) probe. Manual and semi-automated workflows for medium- to high-throughput applications are outlined in this chapter, with western blotting and proteomics-based techniques as the main readouts. This methodology informs us of endogenous deISGylating enzyme expression and activity in a cellular context, including USP18, the type I interferon (IFN-I)-inducible deISGylase, and several constitutively expressed deubiquitinases (DUBs), such as USP5, USP14, USP16, and USP36, that exert cross-reactivity to ISG15. ISG15-ABPP also enables the identification and characterization of potent and selective deISGylating enzyme modulators.
Characterization of ADAMTS9 proteoglycanase activity: comparison with ADAMTS1, ADAMTS4 and ADAMTS5.
A Disintegrin-like And Metalloprotease domain with Thrombospondin type I motifs (ADAMTS) 9 has essential, non-redundant roles during embryogenesis. Adamts9 null murine embryos die prior to completing gastrulation. Unusually for a protease, Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 is required for proteostasis of versican, a widely distributed large aggregating proteoglycan abundant in the provisional extracellular matrix during embryogenesis. Despite its importance, ADAMTS9 proteoglycanase activity has undergone limited characterization, especially in comparison to ADAMTS1, ADAMTS4, and ADAMTS5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, ADAMTS4, and ADAMTS5, ADAMTS9 cleaves versican V1 isoform at E441-A442, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 construct (ADAMTS9 MDTCS) consisting of all ADAMTS 'core domains' present in ADAMTS1, ADAMTS4, and ADAMTS5, and characterized its activity against versican, aggrecan, and the small leucine-rich proteoglycan biglycan. We identified cleavages in versican (V1 and V2 isoforms) and biglycan using a z-score approach based on label-free quantitation of semi- and fully tryptic/GluC peptides. Moreover, using a quantitative assay, we established that ADAMTS9 MDTCS versicanase activity at the E441-A442 site is 175-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 5.5-fold higher than ADAMTS1. Finally, we confirmed that ADAMTS9 MDTCS cleaves bovine aggrecan at E392-A393. This analysis of the proteoglycanase activity in the ADAMTS family highlights differences and similarities in cleavage site specificities which could be leveraged to develop selective small molecule inhibitors against current targets of interest, ADAMTS4, ADAMTS5, and ADAMTS7.
Deep palaeoproteomic profiling of archaeological human brains.
Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.