Prostatic acid phosphatase as a regulator of endo/exocytosis and lysosomal degradation

Tutkimustuotos: OpinnäyteVäitöskirjaArtikkelikokoelma

Abstrakti

Prostatic acid phosphatase (PAP) was discovered during the mid-1930s, but the molecular mechanisms in which this protein is involved remain poorly understood. This enzyme was originally described as a highly-expressed protein in the human prostate that was secreted to the seminal fluid. It has always been associated to prostate cancer, since high levels of acid phosphatase activity were found in the sera of patients with metastatic disease. Therefore, for 40 years, research was focused on the improvement of biochemical assays in an effort to find specific substrates for clinical application. However, in the 1980s PSA (prostate specific antigen) superseded PAP as a biomarker for early detection of the disease and became the preferred marker for diagnosis. Later, in the mid-1990s with the advent of new molecular techniques such as cloning and high-scale protein purification it was possible to obtain high-quality crystals for 3D-structural determinations of PAP. In addition, new concepts emerged concerning the physiological role of the enzyme, with renewed speculation regarding the molecular mechanisms in which this protein could be involved. The fact that the serum levels of the enzyme are increased in prostate cancer patients with metastatic disease rendered this enzyme an attractive target for immunotherapies against advanced prostate cancer. However, this hypothesis has thus far neglected the existence of any potential isoforms that could be expressed in other organs and tissues. Therefore, as part of this thesis, the molecular mechanisms where PAP is involved will be investigated. For this purpose, two biological tools were employed: a PAP-knockout mouse model and stable virus-transfected LNCaP cell lines. A novel transmembrane type-I isoform of PAP (TMPAP) was first characterized as a product of alternative splicing of the same gene (ACPP) that encodes for the well-known secretory isoform (SPAP). TMPAP is distributed throughout mouse tissues, including prostate, lung, kidney, endometrium, salivary glands, and dorsal-root ganglia. The enzyme comprises an N-terminal domain containing the catalytic active site, a transmembrane helical domain, and a short C-terminal cytosolic domain that carries a tyrosine-based motif (Yxxφ) that targets the enzyme to the endosomal-lysosomal/exosomal pathway. This was confirmed by co-localization studies that revealed that PAP localizes to the plasma membrane as well as to the intracellular membranes of vesicles, lysosomes, and intraluminal vesicles of the multivesicular endosomes. Microarray experiments were performed on mouse tissues to study the differential gene expression profile between wild-type and PAP-knockout mice. The differential gene expression that was observed between the prostates of wild-type and PAP-knockout mice suggested that PAP is involved in secretory mechanisms, as many genes related to this process appeared dysregulated. Moreover, the results obtained from two-hybrid system experiments suggested that snapin (a SNARE-associated protein) was a likely candidate protein that could interact with TMPAP. This interaction was recently proved by co-localization and florescence resonance energy transfer (FRET) studies. The PAP-knockout mice developed prostate adenocarcinoma and showed dysregulation of genes related to vesicular traffic. Consequently, this investigation was focused on murine submandibulary glands (SMG) as a model of an exocrine organ. The expression of PAP in SMG was found to be even higher than in mouse prostate. In addition to microarrays and miRNA analyses, physiological and biochemical determinations help to demonstrate that there is an increased salivation volume in PAP-knockout mice upon stimulation with secretagogue drugs. This supports the hypothesis that PAP is involved in the regulation of secretory and exocytic processes. PAP was found to account for 50% of the total acid phosphatase activity in male mouse saliva and it is expressed by the granular convoluted tubular cells of the male SMG but not by the acinar cells. Unlike prostate gland, however, the mouse SMG does not develop signs of hyperplasia or adenocarcinoma in spite of an observed increased acinar cell proliferation. This discrepancy was explained by studying the degree of lymphocyte infiltration, the dysregulation of miRNAs, and the differentially expressed genes in microarray data. In SMGs of PAP-knockout mice, the innate immune system was shown to be responsive and able to remove proliferating acinar cells, which may explain the absence of adenocarcinoma. In addition, the upregulation of anti-inflammatory molecules may prevent the extension of tissue damage. Finally, we compared the effect of the overexpression of SPAP and TMPAP in LNCaP cells with empty-vector cells. As a result, the TMPAP-LNCaP cells exhibited slower growth than SPAP-LNCaP or empty-vector cells. Cells overexpressing either SPAP or TMPAP isoform showed increased 2D-projection area and increased HRP-uptake when compared with empty-vector cells. These two observations suggested an increased vesicular traffic in endo/exocytic pathways to maintain cell membrane homeostasis. Thus, vesicles loaded with TMPAP are most likely sorted to lysosomes by means of its Yxxφ motif. Consequently, there is an increased degradation of cargo molecules such as receptor tyrosine kinases expressed on the cell surface that could explain the observed slow growth of LNCaP cells that overexpress TMPAP. The molecular mechanisms identified in this study will definitely contribute to a better understanding of the physiological role of PAP in diseases and to a critical re-evaluation of existing immunotherapies. The knowledge of the molecular determinants responsible for the presence of TMPAP in the endo/exocytic pathway can also be exploited for the future development of radio-imaging and drug delivery protocols.
Alkuperäiskielienglanti
JulkaisupaikkaHelsinki
Kustantaja
Painoksen ISBN978-951-51-1849-3
Sähköinen ISBN978-951-51-1850-9
TilaJulkaistu - 29 tammik. 2016
OKM-julkaisutyyppiG5 Tohtorinväitöskirja (artikkeli)

Tieteenalat

  • 1182 Biokemia, solu- ja molekyylibiologia

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