The human deubiquitinating enzyme ubiquitin-specific protease 2 (USP2) regulates multiple cellular pathways, including cell proliferation and apoptosis. As a result of alternative splicing four USP2 isoenzymes are expressed in human cells of which all contain a weak peroxisome targeting signal of type 1 (PTS1) at their C-termini. Here, we systematically analyzed apoptotic effects induced by overexpression and intracellular localization for each isoform. All isoforms exhibit proapoptotic activity and are post-translationally imported into the matrix of peroxisomes in a PEX5-dependent manner. However, a significant fraction of the USP2 pool resides in the cytosol due to a weaker PTS1 and thus low affinity to the PTS receptor PEX5. Blocking of peroxisomal import did not interfere with the proapoptotic activity of USP2, suggesting that the enzyme performs its critical function outside of this compartment. Instead, increase of the efficiency of USP2 import into peroxisomes either by optimization of its peroxisomal targeting signal or by overexpression of the PTS1 receptor did result in a reduction of the apoptotic rate of transfected cells. Our studies suggest that peroxisomal import of USP2 provides additional control over the proapoptotic activity of cytosolic USP2 by spatial separation of the deubiquitinating enzymes from their interaction partners in the cytosol and nucleus.

Female pattern hair loss (FPHL) is a non-scarring alopecia resulting from the progressive conversion of the terminal (t) scalp hair follicles (HFs) into intermediate/miniaturized (i/m) HFs. Although data supporting nutrient deficiency in FPHL HFs are lacking, therapeutic strategies are often associated with nutritional supplementation. Here, we show by metabolic analysis that selected nutrients important for hair growth such as essential amino acids and vitamins are indeed decreased in affected iHFs compared to tHFs in FPHL scalp skin, confirming nutrient insufficiency. iHFs also displayed a more quiescent metabolic phenotype, as indicated by altered metabolite abundance in freshly collected HFs and release/consumption during organ culture of products/substrates of TCA cycle, aerobic glycolysis, and glutaminolysis. Yet, as assessed by exogenous nutrient supplementation ex vivo, nutrient uptake mechanisms are not impaired in affected FPHL iHFs. Moreover, blood vessel density is not diminished in iHFs versus tHFs, despite differences in tHFs from different FPHL scalp locations or versus healthy scalp or changes in the expression of angiogenesis-associated growth factors. Thus, our data reveal that affected iHFs in FPHL display a relative nutrient insufficiency and dormant metabolism, but are still capable of absorbing nutrients, supporting the potential of nutritional supplementation as an adjunct therapy for FPHL.

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Vitiligo is an autoimmune disease of the skin in which CD8+ T cells kill melanocytes, leading to skin depigmentation. The IFNg-dependent chemokines CXCL9 and CXCL10 are highly expressed in human vitiligo lesions, and CXCL10 is required for depigmentation in our mouse model of vitiligo. New drugs are being developed to target this pathway as a novel treatment strategy. In order to further characterize the immune infiltrate in vitiligo lesions and improve our ability to quantify disease activity for future clinical trials, we adapted a suction blistering technique to access skin interstitial fluid for protein analysis by ELISA and cell immunophenotyping by flow cytometry. Using separate groups to first define and later validate candidate biomarkers, we observed that CD8+ T cell number and CXCL9 protein concentration were consistently elevated in lesional skin, but not in non-lesional skin or healthy subjects. Surprisingly, CXCL10 protein was not reliably detected in lesional skin. Phenotyping of the T cells in the skin interstitial fluid revealed that nearly all CD8+ T cells in the skin express both CXCR3, the chemokine receptor for CXCL9 and CXCL10, and PD1, an immune checkpoint inhibitor. While the absolute number of T regulatory cells (Tregs) in the skin was the same between lesional and non-lesional skin, the ratio of CD8+ T cells to Tregs was elevated in active vitiligo lesions, suggesting that this ratio is key to modulating inflammation and tolerance within vitiligo lesions. These results suggest that CD8+ T cell number and CXCL9 protein concentration in the skin may serve as biomarkers of active disease and treatment response, and that the suction blistering technique can provide new insight into the immune infiltrate in vitiligo.

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