Dysregulated Cerebrospinal Fluid Proteome of Spinocerebellar Ataxia Type 2 and its Clinical Implications.

BACKGROUND: Abnormalities in ataxin-2 associated with spinocerebellar ataxia type 2 (SCA2) may lead to widespread disruptions in the proteome. This study was performed to identify dysregulated proteome in SCA2 and to explore its clinical-radiological correlations. METHODS: Cerebrospinal fluid (CSF) samples from 21 genetically confirmed SCA2 were subjected to shotgun proteome analysis using mass spectrometry (MS) and tandem mass tag (TMT)-based multiplexing. Proteins with at least 1.5-fold change in abundance were identified. Their relative abundance was measured using parallel reaction monitoring (PRM) and correlated against disease-related factors. RESULTS: Eleven proteins were significantly upregulated in SCA2. They belonged to the family of cell adhesion molecules and granins. Their fold changes showed significant clinical, genetic, and radiological correlations. CONCLUSIONS: Significant dysregulation of CSF proteome is seen in SCA2. The dysregulated protein may have potential use in clinical evaluation of patients with SCA2. © 2024 International Parkinson and Movement Disorder Society.

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma.

Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Recent progress in mass spectrometry-based urinary proteomics.

Serum or plasma is frequently utilized in biomedical research; however, its application is impeded by the requirement for invasive sample collection. The non-invasive nature of urine collection makes it an attractive alternative for disease characterization and biomarker discovery. Mass spectrometry-based protein profiling of urine has led to the discovery of several disease-associated biomarkers. Proteomic analysis of urine has not only been applied to disorders of the kidney and urinary bladder but also to conditions affecting distant organs because proteins excreted in the urine originate from multiple organs. This review provides a progress update on urinary proteomics carried out over the past decade. Studies summarized in this review have expanded the catalog of proteins detected in the urine in a variety of clinical conditions. The wide range of applications of urine analysis-from characterizing diseases to discovering predictive, diagnostic and prognostic markers-continues to drive investigations of the urinary proteome.

Neuroanatomical zones of human traumatic brain injury reveal significant differences in protein profile and protein oxidation: Implications for secondary injury events.

Traumatic brain injury (TBI) causes significant neurological deficits and long-term degenerative changes. Primary injury in TBI entails distinct neuroanatomical zones, i.e., contusion (Ct) and pericontusion (PC). Their dynamic expansion could contribute to unpredictable neurological deterioration in patients. Molecular characterization of these zones compared with away from contusion (AC) zone is invaluable for TBI management. Using proteomics-based approach, we were able to distinguish Ct, PC and AC zones in human TBI brains. Ct was associated with structural changes (blood-brain barrier (BBB) disruption, neuroinflammation, axonal injury, demyelination and ferroptosis), while PC was associated with initial events of secondary injury (glutamate excitotoxicity, glial activation, accumulation of cytoskeleton proteins, oxidative stress, endocytosis) and AC displayed mitochondrial dysfunction that could contribute to secondary injury events and trigger long-term degenerative changes. Phosphoproteome analysis in these zones revealed that certain differentially phosphorylated proteins synergistically contribute to the injury events along with the differentially expressed proteins. Non-synaptic mitochondria (ns-mito) was associated with relatively more differentially expressed proteins (DEPs) compared to synaptosomes (Syn), while the latter displayed increased protein oxidation including tryptophan (Trp) oxidation. Proteomic analysis of immunocaptured complex I (CI) from Syn revealed increased Trp oxidation in Ct > PC > AC (vs. control). Oxidized W272 in the ND1 subunit of CI, revealed local conformational changes in ND1 and the neighboring subunits, as indicated by molecular dynamics simulation (MDS). Taken together, neuroanatomical zones in TBI show distinct protein profile and protein oxidation representing different primary and secondary injury events with potential implications for TBI pathology and neurological status of the patients.

Tissue proteome analysis for profiling proteins associated with lymph node metastasis in gallbladder cancer.

Lymph node (LN) metastasis is the earliest sign of metastatic spread and an established predictor of poor outcome in gallbladder cancer (GBC). Patients with LN positive GBC have a significantly worse survival (median survival- 7 months) than patients with LN negative disease (median survival- ~ 23 months) in spite of standard treatment which includes extended surgery followed by chemotherapy, radiotherapy and targeted therapy. This study aims at understanding the underlying molecular processes associated with LN metastasis in GBC. Here, we used iTRAQ-based quantitative proteomic analysis using tissue cohort comprising of primary tumor of LN negative GBC (n = 3), LN positive GBC (n = 4) and non-tumor controls (Gallstone disease, n = 4), to identify proteins associated with LN metastasis. A total of 58 differentially expressed proteins (DEPs) were found to be specifically associated with LN positive GBC based on the criteria of p value ≤ 0.05, fold change ≥ 2 and unique peptides ≥ 2. These include the cytoskeleton and associated proteins such as keratin, type II cytoskeletal 7 (KRT7), keratin type I cytoskeletal 19 (KRT19), vimentin (VIM), sorcin (SRI) and nuclear proteins such as nucleophosmin Isoform 1 (NPM1), heterogeneous nuclear ribonucleoproteins A2/B1 isoform X1 (HNRNPA2B1). Some of them are reported to be involved in promoting cell invasion and metastasis. Bioinformatic analysis of the deregulated proteins in LN positive GBC using STRING database identified 'neutrophil degranulation' and 'HIF1 activation' to be among the top deregulated pathways. Western blot and IHC analysis showed a significant overexpression of KRT7 and SRI in LN positive GBC in comparison to LN negative GBC. KRT7, SRI and other proteins may be further explored for their diagnostics and therapeutic applications in LN positive GBC.

Mitochondrial Complex I Inhibition in Dopaminergic Neurons Causes Altered Protein Profile and Protein Oxidation: Implications for Parkinson's disease.

Mitochondrial dysfunction and oxidative stress are critical to neurodegeneration in Parkinson's disease (PD). Mitochondrial dysfunction in PD entails inhibition of the mitochondrial complex I (CI) in the dopaminergic neurons of substantia nigra. The events contributing to CI inhibition and downstream pathways are not completely elucidated. We conducted proteomic analysis in a dopaminergic neuronal cell line exposed individually to neurotoxic CI inhibitors: rotenone (Rot), paraquat (Pq) and 1-methyl-4-phenylpyridinium (MPP+). Mass spectrometry (MS) revealed the involvement of biological processes including cell death pathways, structural changes and metabolic processes among others, most of which were common across all models. The proteomic changes induced by Pq were significantly higher than those induced by Rot and MPP+. Altered metabolic processes included downregulated mitochondrial proteins such as CI subunits. MS of CI isolated from the models revealed oxidative post-translational modifications with Tryptophan (Trp) oxidation as the predominant modification. Further, 62 peptides in 22 subunits of CI revealed Trp oxidation with 16 subunits common across toxins. NDUFV1 subunit had the greatest number of oxidized Trp and Rot model displayed the highest number of Trp oxidation events compared to the other models. Molecular dynamics simulation (MDS) of NDUFV1 revealed that oxidized Trp 433 altered the local conformation thereby changing the distance between the Fe-S clusters, Fe-S 301(N1a) to Fe-S 502 (N3) and Fe-S 802 (N4) to Fe-S 801 (N5), potentially affecting the efficiency of electron transfer. The events triggered by the neurotoxins represent CI damage, mitochondrial dysfunction and neurodegeneration in PD.

Integrated proteomics and phosphoproteomics revealed druggable kinases in neoadjuvant chemotherapy resistant tongue cancer.

Tongue squamous cell carcinoma is an aggressive oral cancer with a high incidence of metastasis and poor prognosis. Most of the oral cavity cancer patients present in clinics with locally advanced unresectable tumors. Neoadjuvant treatment is beneficial for these individuals as it reduces the tumor size aiding complete resection. However, patients develop therapy resistance to the drug regimen. In this study, we explored the differential expression of proteins and altered phosphorylation in the neoadjuvant chemotherapy resistant tongue cancer patients. We integrated the proteomic and phosphoproteomic profiles of resistant (n = 4) and sensitive cohorts (n = 4) and demonstrated the differential expression and phosphorylation of proteins in the primary tissue of the respective subject groups. We observed differential and extensive phosphorylation of keratins such as KRT10 and KRT1 between the two cohorts. Furthermore, our study revealed a kinase signature associated with neoadjuvant chemotherapy resistance. Kinases such as MAPK1, AKT1, and MAPK3 are predicted to regulate the resistance in non-responders. Pathway analysis showed enrichment of Rho GTPase signaling and hyperphosphosphorylation of proteins involved in cell motility, invasion, and drug resistance. Targeting the kinases could help with the clinical management of neoadjuvant chemotherapy-resistant tongue cancer.

Phosphoprotein network analysis of corneal epithelium of keratoconus patients.

Keratoconus (KC) is non-inflammatory, bilateral progressive corneal ectasia, and a disease of established biomechanical instability. The etiology of KC is believed to be multifactorial. Although previous studies gained insight into the understanding of the disease, little is known thus far on global protein phosphorylation changes in keratoconus. We performed phosphoproteome analysis of corneal epithelium from control (N = 5) and KC patients. Tandem mass tag (TMT) multiplexing technology along with immobilized metal affinity chromatography (IMAC) were used for the phosphopeptides enrichment and quantitation. Enriched peptides were analyzed on Orbitrap Fusion Tribrid mass spectrometer. This leads to the identification of 2939 unique phosphopeptides derived from 1270 proteins. We observed significant differential phosphorylation of 591 phosphopeptides corresponding to 375 proteins. Our results provide first phosphoproteomic signature of the keratoconus disease and identified dysregulated signaling pathways that can be targeted for therapy in future studies.

Quantitative tissue proteome profile reveals neutrophil degranulation and remodeling of extracellular matrix proteins in early stage gallbladder cancer.

Gallbladder cancer (GBC) is an aggressive malignancy of the gastrointestinal tract with a poor prognosis. It is important to understand the molecular processes associated with the pathogenesis of early stage GBC and identify proteins useful for diagnostic and therapeutic strategies. Here, we have carried out an iTRAQ-based quantitative proteomic analysis of tumor tissues from early stage GBC cases (stage I, n=7 and stage II, n=5) and non-tumor controls (n=6) from gallstone disease (GSD). We identified 357 differentially expressed proteins (DEPs) based on ≥ 2 unique peptides and ≥ 2 fold change with p value < 0.05. Pathway analysis using the STRING database showed, 'neutrophil degranulation' to be the major upregulated pathway that includes proteins such as MPO, PRTN3, S100A8, MMP9, DEFA1, AZU, and 'ECM organization' to be the major downregulated pathway that includes proteins such as COL14A1, COL1A2, COL6A1, COL6A2, COL6A3, BGN, DCN. Western blot and/or IHC analysis confirmed the elevated expression of MPO, PRTN3 and S100A8 in early stage of the disease. Based on the above results, we hypothesize that there is an increased neutrophil infiltration in tumor tissue and neutrophil degranulation leading to degradation of extracellular matrix (ECM) proteins promoting cancer cell invasion in the early stage GBC. Some of the proteins (MPO, MMP9, DEFA1) associated with 'neutrophil degranulation' showed the presence of 'signal sequence' suggesting their potential as circulatory markers for early detection of GBC. Overall, the study presents a protein dataset associated with early stage GBC.

Graphitic Carbon Nitride Causes Widespread Global Molecular Changes in Epithelial and Fibroblast Cells.

For scaffold and imaging applications, nanomaterials such as graphene and its derivatives have been widely used. Graphitic carbon nitride (g-C3N4) is among one such derivative of graphenes, which draws strong consideration due to its physicochemical properties and photocatalytic activity. To use g-C3N4 for biological applications, such as molecular imaging or drug delivery, it must interact with the epithelium, cross the epithelial barrier, and then come in contact with the extracellular matrix of the fibroblast cells. Thus, it becomes essential to understand its molecular mechanism of action. Hence, in this study, to understand the molecular reprogramming associated with g-C3N4, global gene expression using DNA microarrays and proteomics using tandem mass tag (TMT) labeling and mass spectrometry were performed in epithelial and fibroblast cells, respectively. Our results showed that g-C3N4 can cross the epithelial barrier by regulating the adherens junction proteins. Further, using g-C3N4-PDMS scaffolds as a mimic of the extracellular matrix for fibroblast cells, the common signaling pathways were identified between the epithelium and fibroblast cells. These pathways include Wnt signaling, integrin signaling, TGF-ß signaling, cadherin signaling, oxidative stress response, ubiquitin proteasome pathway, and EGF receptor signaling pathways. These altered signature pathways identified could play a prominent role in g-C3N4-mediated cellular interactions in both epithelial and fibroblast cells. Additionally, ß catenin, EGFR, and MAP2K2 protein-protein interaction networks could play a prominent role in fibroblast cell proliferation. The findings could further our knowledge on g-C3N4-mediated alterations in cellular molecular signatures, enabling the potential use of these materials for biological applications such as molecular imaging and drug delivery.