CD6 triggers actomyosin cytoskeleton remodeling after binding to its receptor complex.

The T cell marker CD6 regulates both T cells and target cells during inflammatory responses by interacting with its receptors. However, only a few receptors binding to the extracellular domains of CD6 have been identified, and cellular events induced by CD6 engagement with its receptors in target cells remain poorly understood. In this study, we identified CD44 as a novel CD6 receptor by proximity labeling and confirmed the new CD6-CD44 interaction by biochemical and biophysical approaches. CD44 and the other 2 known CD6 receptors, CD166 and CDCP1, were distributed diffusely on resting retinal pigment epithelium (RPE) cells but clustered together to form a receptor complex upon CD6 binding. CD6 stimulation induced dramatic remodeling of the actomyosin cytoskeleton in RPE cells mediated by activation of RhoA, and Rho-associated kinase signaling, resulting in increased myosin II phosphorylation. Such actomyosin activation triggered the disassembly of tight junctions responsible for RPE barrier integrity in a process that required all components of the tripartite CD6 receptor complex. These data provided new insights into the mechanisms by which CD6 mediates T cell-driven disruption of tissue barriers during inflammation.

Proteomics of Primary Uveal Melanoma: Insights into Metastasis and Protein Biomarkers.

Uveal melanoma metastases are lethal and remain incurable. A quantitative proteomic analysis of 53 metastasizing and 47 non-metastasizing primary uveal melanoma (pUM) was pursued for insights into UM metastasis and protein biomarkers. The metastatic status of the pUM specimens was defined based on clinical data, survival histories, prognostic analyses, and liver histopathology. LC MS/MS iTRAQ technology, the Mascot search engine, and the UniProt human database were used to identify and quantify pUM proteins relative to the normal choroid excised from UM donor eyes. The determined proteomes of all 100 tumors were very similar, encompassing a total of 3935 pUM proteins. Proteins differentially expressed (DE) between metastasizing and non-metastasizing pUM (n = 402) were employed in bioinformatic analyses that predicted significant differences in the immune system between metastasizing and non-metastasizing pUM. The immune proteins (n = 778) identified in this study support the immune-suppressive nature and low abundance of immune checkpoint regulators in pUM, and suggest CDH1, HLA-DPA1, and several DE immune kinases and phosphatases as possible candidates for immune therapy checkpoint blockade. Prediction modeling identified 32 proteins capable of predicting metastasizing versus non-metastasizing pUM with 93% discriminatory accuracy, supporting the potential for protein-based prognostic methods for detecting UM metastasis.

Disease-specific platelet signaling defects in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a rapidly progressive disease with several treatment options. Long-term mortality remains high with great heterogeneity in treatment response. Even though most of the pathology of IPAH is observed in the lung, there is systemic involvement. Platelets from patients with IPAH have characteristic metabolic shifts and defects in activation; therefore, we investigated whether they could be used to identify other disease-specific abnormalities. We used proteomics to investigate protein expression changes in platelets from patients with IPAH compared with healthy controls. Key abnormalities of nitric oxide pathway were tested in platelets from a larger cohort of unique patients with IPAH. Platelets showed abnormalities in the prostacyclin and nitric oxide pathways, which are dysregulated in IPAH and hence targets of therapy. We detected reduced expression of G protein αs and increased expression of the regulatory subunits of the cAMP-dependent protein kinase (PKA) type II isoforms, supporting an overall decrease in the activation of the prostacyclin pathway. We noted reduced levels of the soluble guanylate cyclase (sGC) subunits and increased expression of the phosphodiesterase type 5 A (PDE5A), conditions that affect the response to nitric oxide. Ensuing analysis of 38 unique patients with IPAH demonstrated considerable variation in the levels and specific activity of sGC, a finding with novel implications for personalized therapy. Platelets have some of the characteristic vasoactive signal abnormalities seen in IPAH and may provide comprehensive ex vivo mechanistic information to direct therapeutic decisions.

Quantitative proteomic comparison of myofibroblasts derived from bone marrow and cornea.

Myofibroblasts are fibroblastic cells that function in wound healing, tissue repair and fibrosis, and arise from bone marrow (BM)-derived fibrocytes and a variety of local progenitor cells. In the cornea, myofibroblasts are derived primarily from stromal keratocytes and from BM-derived fibrocytes after epithelial-stromal and endothelial-stromal injuries. Quantitative proteomic comparison of mature alpha-smooth muscle actin (α-SMA)+ myofibroblasts (verified by immunocytochemistry for vimentin, α-SMA, desmin, and vinculin) generated from rabbit corneal fibroblasts treated with transforming growth factor (TGF) beta-1 or generated directly from cultured BM treated with TGF beta-1 was pursued for insights into possible functional differences. Paired cornea-derived and BM-derived α-SMA+ myofibroblast primary cultures were generated from four New Zealand white rabbits and confirmed to be myofibroblasts by immunocytochemistry. Paired cornea- and BM-derived myofibroblast specimens from each rabbit were analyzed by LC MS/MS iTRAQ technology using an Orbitrap Fusion Lumos Tribrid mass spectrometer, the Mascot search engine, the weighted average quantification method and the UniProt rabbit and human databases. From 2329 proteins quantified with ≥ 2 unique peptides from ≥ 3 rabbits, a total of 673 differentially expressed (DE) proteins were identified. Bioinformatic analysis of DE proteins with Ingenuity Pathway Analysis implicate progenitor-dependent functional differences in myofibroblasts that could impact tissue development. Our results suggest BM-derived myofibroblasts may be more prone to the formation of excessive cellular and extracellular material that are characteristic of fibrosis.