Procathepsin V Is Secreted in a TSH Regulated Manner from Human Thyroid Epithelial Cells and Is Accessible to an Activity-Based Probe

Abstract

The significance of cysteine cathepsins for the liberation of thyroid hormones from the precursor thyroglobulin was previously shown by in vivo and in vitro studies. Cathepsin L is most important for thyroglobulin processing in mice. The present study aims at specifying the possible contribution of its closest relative, cysteine cathepsin L2/V, to thyroid function. Immunofluorescence analysis on normal human thyroid tissue revealed its predominant localization at the apical plasma membrane of thyrocytes and within the follicle lumen, indicating the secretion of cathepsin V and extracellular tasks rather than its acting within endo-lysosomes. To explore the trafficking pathways of cathepsin V in more detail, a chimeric protein consisting of human cathepsin V tagged with green fluorescent protein (GFP) was stably expressed in the Nthy-ori 3-1 thyroid epithelial cell line. Colocalization studies with compartment-specific markers and analyses of post-translational modifications revealed that the chimeric protein was sorted into the lumen of the endoplasmic reticulum and subsequently transported to the Golgi apparatus, while being N-glycosylated. Immunoblotting showed that the chimeric protein reached endo-lysosomes and it became secreted from the transduced cells. Astonishingly, thyroid stimulating hormone (TSH)-induced secretion of GFP-tagged cathepsin V occurred as the proform, suggesting that TSH upregulates its transport to the plasma membrane before it reaches endo-lysosomes for maturation. The proform of cathepsin V was found to be reactive with the activity-based probe DCG-04, suggesting that it possesses catalytic activity. We propose that TSH-stimulated secretion of procathepsin V is the default pathway in the thyroid to enable its contribution to thyroglobulin processing by extracellular means.

Investigations on Primary Cilia of Nthy-ori 3-1 Cells upon Cysteine Cathepsin Inhibition or Thyrotropin Stimulation

“…This is in line with the previous observation of the TSH-regulated expression of cathepsin S in the embryonic development of rats [28]. Furthermore, our group previously found the proform of cathepsin L2/V to be secreted and extracellularly matured in Nthy-ori 3-1 cells stably expressing a green fluorescent protein-tagged form [29]. Moreover, procathepsin L2 was reactive with the ABP DCG-04, suggesting that it possesses catalytic activity [29].…”

“…Furthermore, our group previously found the proform of cathepsin L2/V to be secreted and extracellularly matured in Nthy-ori 3-1 cells stably expressing a green fluorescent protein-tagged form [29]. Moreover, procathepsin L2 was reactive with the ABP DCG-04, suggesting that it possesses catalytic activity [29]. Therefore, the decrease of cilia length could also be due to the increase of cysteine cathepsin activity, even irrespective of the presence of mature forms, as initially hypothesized.…”

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity

“…Elevated cathepsin V levels have been associated with various pathological processes, including myasthenia gravis [9] , atherosclerosis [8] , vascular inflammation [14] , type 1 diabetes [15] , mucopolysaccharidoses [16] , neurological diseases [12] , and cancer [3] . In cancer, high cathepsin V expression was first detected in colorectal and breast carcinomas [3] , and later also in ovarian, endometrial, renal, squamous cell, thymic epithelial [17] , hepatocellular, and thyroid carcinomas [18] , [19] . Higher cathepsin V levels were associated with increasing tumor stage [20] , [21] , distant metastases [22] , [23] , [24] , and lower patient survival [24] .…”

Cathepsin V regulates cell cycle progression and histone stability in the nucleus of breast cancer cells

“…Frontiers in Pharmacology frontiersin.org the nuclear compartment including the regulation of cell proliferation, transcription factor and histone processing (Goulet et al, 2004;Goulet et al, 2007;Duncan et al, 2008;Khalkhali-Ellis et al, 2014;Bach et al, 2015;Tamhane et al, 2016;Al-Hashimi et al, 2020b). Previous research has also documented interactions between the histones and cathepsins, beyond proteolytic cleavage.…”