Orosco, A. | Search bioRxiv

bioRxiv2024

Keratin 16 spatially inhibits type I interferon responses in stressed skin

The stress-induced keratin 16 is broadly used as a biomarker in inflammatory skin disorders while pathogenic variants in KRT16 cause pachyonychia congenita (PC), a condition in which differentiation and homeostasis are disrupted in palmoplantar epidermis and epithelial appendages. How K16 impacts these disorders at a molecular level is poorly understood. Here we report that K16 spatially restricts type I interferon (IFN) signaling and innate immunity in palmoplantar keratoderma (PPK) lesions in PC patients, imiquimod- and phorbol ester-induced models of sterile inflammation in mouse skin, and poly(I:C)-treated human keratinocytes ex vivo. Mechanistically, K16 interacts with effectors of the RIG-I-like receptor (RLR) pathway, including 14-3-3{varepsilon}, and inhibits the 14-3-3{varepsilon}:RIG-I interaction upstream of IFN activation. Topical application of the JAK inhibitor Ruxolitinib reduces the severity of PC-PPK-like lesions in Krt16 null mice. These findings uncover a new paradigm for keratin-dependent regulation of innate immunity and suggest a new approach to PC treatment. One sentence summaryKRT16 negatively regulates type I interferon signaling and innate immune responses in the skin, offering insight into the pathophysiology of inflammatory skin diseases including pachyonychia congenita, psoriasis and others.

immunology↗

bioRxiv2019

MitoPlex: A Targeted Multiple Reaction Monitoring Assay for Quantification of a Curated Set of Mitochondrial Proteins

Mitochondria are the major source of cellular energy (ATP), as well as critical mediators of widespread functions such as cellular redox balance, apoptosis, and metabolic flux. Methods to quantify mitochondrial content are limited to low throughput immunoassays, measurement of mitochondrial DNA, or relative quantification by untargeted mass spectrometry. Here, we present a high throughput, reproducible and quantitative mass spectrometry multiple reaction monitoring based assay of 37 proteins critical to central carbon chain metabolism and overall mitochondrial function termed MitoPlex. We coupled this protein multiplex with a parallel analysis of the central carbon chain metabolites (218 metabolite assay) extracted in tandem from the same sample, be it cells or tissue. In tests of its biological applicability in cells and tissues, MitoPlex plus metabolites indicated profound effects of HMG-CoA Reductase inhibition (e.g., statin treatment) on mitochondria of i) differentiating C2C12 skeletal myoblasts, as well as a clear opposite trend of statins to promote mitochondrial protein expression and metabolism in heart and liver, while suppressing mitochondrial protein and ii) aspects of metabolism in the skeletal muscle obtained from C57Bl6 mice. Our results not only reveal new insights into the metabolic effect of statins in skeletal muscle, but present a new high throughput, reliable MS-based tool to study mitochondrial dynamics in both cell culture and in vivo models.

molecular biology↗