Different Mutation Tolerance of Lentiviral (HIV-1) and Deltaretroviral (BLV and HTLV) Protease Precursors.

The bovine leukemia virus (BLV) and the human T-lymphothropic viruses (HTLVs) are members of the deltaretrovirus genus of Retroviridae family. An essential event of the retroviral life cycle is the processing of the polyproteins by the viral protease (PR); consequently, these enzymes became important therapeutic targets of the anti-retroviral drugs. As compared to human immunodeficiency viruses (HIVs), the deltaretroviruses have a different replication strategy, as they replicate predominantly in the DNA form, by forcing the infected cell to divide, unlike HIV-1, which replicates mainly by producing a vast number of progeny virions and by reinfection. Due to bypassing the error-prone reverse transcription step of replication, the PRs of deltaretroviruses did not undergo such extensive evolution as HIV PRs and remained more highly conserved. In this work, we studied the abilities of wild-type and modified BLV, HTLV (type 1, 2 and 3), and HIV-1 PRs (fused to an N-terminal MBP tag) for self-processing. We designed a cleavage site mutant MBP-fused BLV PR precursor as well, this recombinant enzyme was unable for self-proteolysis, the MBP fusion tag decreased its catalytic efficiency but showed an unusually low Ki for the IB-268 protease inhibitor. Our results show that the HTLV and BLV deltaretrovirus PRs exhibit lower mutation tolerance as compared to HIV-1 PR, and are less likely to retain their activity upon point mutations at various positions, indicating a higher flexibility of HIV-1 PR in tolerating mutations under selective pressure.

Cigarette smoke toxin hydroquinone and misfolding pancreatic lipase variant cooperatively promote endoplasmic reticulum stress and cell death.

Mutation-induced protein misfolding of pancreatic secretory enzymes and consequent endoplasmic reticulum stress can cause chronic pancreatitis. A recent study revealed that cigarette smoke also increases the risk of the disease through endoplasmic reticulum stress. Here, we investigated the cumulative cellular effect of the G233E misfolding human pancreatic lipase variant and hydroquinone; a main toxic constituent of cigarette smoke, using mammalian cell lines. We found that hydroquinone reduces cell viability on a dose-dependent manner through programmed cell death, and diminishes lipase secretion without affecting its expression. Interestingly, hydroquinone decreased the viability more markedly in cells expressing the G233E lipase variant, than in cells producing wild-type lipase. The more substantial viability loss was due to increased endoplasmic reticulum stress, as demonstrated by elevated levels of X-box binding protein 1 mRNA splicing and immunoglobulin binding protein, NAD(P)H:quinone oxidoreductase 1 and C/EBP homologous protein expression. Unresolved endoplasmic reticulum stress, and especially up-regulation of the pro-apoptotic transcription factor C/EBP homologous protein were likely responsible for the increased cell death. Our observations demonstrated that the combination of hydroquinone and misfolding pancreatic lipase variant promote increased levels of endoplasmic reticulum stress and cell death, which may predispose to chronic pancreatitis.

Missense PNLIP mutations impeding pancreatic lipase secretion cause protein misfolding and endoplasmic reticulum stress.

BACKGROUND/OBJECTIVE: Mutation-induced misfolding of digestive enzymes has been shown to cause chronic pancreatitis. Recently, heterozygous pancreatic lipase (PNLIP) mutations leading to reduced secretion were identified. The aim of the present study was to investigate whether PNLIP mutants with a secretion defect result in endoplasmic reticulum (ER) stress in cell culture models. METHODS: We introduced the coding DNA for wild-type and A174P, G233E, C254R and V454F mutant PNLIP into two mammalian cell lines and carried out functional assays to assess PNLIP expression, secretion and ER stress. RESULTS: We found that wild-type PNLIP was readily secreted from the investigated cell lines. In contrast, none of the lipase mutants were detectable in the conditioned media. PNLIP variants accumulated in the cells as intracellular protein aggregates probably due to misfolding in the ER. Consistent with this notion, PNLIP mutants induced ER stress, as indicated by increased mRNA levels of spliced X-box Binding Protein 1 (XBP1) and the ER chaperone Immunoglobulin Binding Protein (BiP). CONCLUSION: The results indicate that PNLIP mutations associated with a lipase secretion defect cause ER stress and thereby may increase the risk for chronic pancreatitis.

Biochemical Characterization, Specificity and Inhibition Studies of HTLV-1, HTLV-2, and HTLV-3 Proteases.

The human T-lymphotropic viruses (HTLVs) are causative agents of severe diseases including adult T-cell leukemia. Similar to human immunodeficiency viruses (HIVs), the viral protease (PR) plays a crucial role in the viral life-cycle via the processing of the viral polyproteins. Thus, it is a potential target of anti-retroviral therapies. In this study, we performed in vitro comparative analysis of human T-cell leukemia virus type 1, 2, and 3 (HTLV-1, -2, and -3) proteases. Amino acid preferences of S4 to S1' subsites were studied by using a series of synthetic oligopeptide substrates representing the natural and modified cleavage site sequences of the proteases. Biochemical characteristics of the different PRs were also determined, including catalytic efficiencies and dependence of activity on pH, temperature, and ionic strength. We investigated the effects of different HIV-1 PR inhibitors (atazanavir, darunavir, DMP-323, indinavir, ritonavir, and saquinavir) on enzyme activities, and inhibitory potentials of IB-268 and IB-269 inhibitors that were previously designed against HTLV-1 PR. Comparative biochemical analysis of HTLV-1, -2, and -3 PRs may help understand the characteristic similarities and differences between these enzymes in order to estimate the potential of the appearance of drug-resistance against specific HTLV-1 PR inhibitors.