To examine the overall degradation rate of the PC variants the cells were washed twice with methionine-deficient medium and cultured for 30 min in methionine-deficient medium containing 50 Ci/ml [35S]methionine (Perkin Elmer, Boston, MA, USA). protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR Rabbit Polyclonal to Tau (phospho-Ser516/199) activation demonstrated by increased expression levels of the ER stress PD318088 markers BiP and P-eIF2 and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation. Conclusions/Significance The reduced intracellular level and impaired secretion PD318088 of the PC mutant were due PD318088 to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis. Introduction Activated protein C (PC) is a vitamin K-dependent plasma glycoprotein that plays an important role in the regulation of blood coagulation [1]. PC deficiency is caused by mutations in the gene encoding PC, and is clinically associated with increased risk of venous thrombosis [2]. At present, nearly 200 various mutations in the PC gene have been described [3] and the functional effects of several PC mutations shown to be associated with PC deficiency have previously been studied em in-vitro /em [4]C[11]. The majority of the missense mutations in PC lead to protein misfolding and consequently to retention of the mutants in the endoplasmic reticulum (ER) with subsequent degradation by proteasomes in a process called ER associated degradation (ERAD) [11]C[13]. PC PD318088 is synthesized in liver cells where it is subjected to several posttranslational modifications in the ER and in the Golgi apparatus [14]. The processing of proteins in ER is controlled by chaperones, which facilitate protein folding and ensure that only correctly folded proteins are transported from the ER to Golgi [15]. Accumulation of misfolded proteins in ER can disturb homeostasis and result in ER stress, which activates the cellular unfolded protein response (UPR). This response eliminates ER stress by increasing the efficiency of protein folding, promoting ERAD and attenuating protein synthesis of mutated proteins [16]. Up-regulated expression of chaperones has been demonstrated in several studies on mutated proteins in general [17], [18]. A majority of the reported misfolded glycoproteins, including some mutated PC variants [11]C[13], are retrotranslocated across the ER membrane and degraded by ERAD. Some of the other mutant proteins are degraded by other proteases found in the ER and in the cytosol [19]C[21]. However, a few studies have described misfolded proteins, which were retained in the ER for a longer period of time without being degraded at all. These proteins were accumulated in the ER and led to elevated ER stress evidenced by increased expression levels of proteins such as the immunoglobulin-binding protein (BiP), the protein kinase-like ER kinase (PERK), and the CCAAT/enhancer-binding protein homologous protein (CHOP), all common markers of ER stress and UPR activation [17], [18], [22]. It has been shown that accumulation of misfolded proteins in the ER was associated with activation of PERK resulting in phosphorylation of the eukaryotic initiation factor 2 (eIF2) with subsequent down-regulation of the protein synthesis [22]C[24]. Prolonged ER stress and UPR activation are associated with ERAD dysfunction, cell injury and apoptosis contributing to pathogenesis of many diseases [17], [22], [23], [25]C[27]. In a recent study [28], we found that both the intra- and extracellular levels of PD318088 the PC-A267T mutant were strongly reduced compared to the.
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