HIV-1 protease-induced apoptosis

Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, Tomáš

doi:10.1186/1742-4690-11-37

Cited by 41 publications

(48 citation statements)

References 62 publications

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“…To independently confirm the reported RIPK2-PR interaction, we over-expressed and affinity purified (AP) epitope tagged proteins in HEK293T cells. Since over-expression of wild-type PR is cytotoxic [ 44 – 46 ], a catalytically inactive PR mutant (D25 N) was used in our studies. We observed binding of PR (D25 N) to RIPK2.…”

Section: Resultsmentioning

confidence: 99%

HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2

2015

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BackgroundHIV-1 protease (PR) is essential for viral infectivity as it cleaves Gag and Gag-Pol polyprotein precursors during viral maturation. Recent evidence suggests that cellular proteins can also be cleaved by PR, perhaps representing an important viral strategy to counter host defense mechanisms. Receptor-interacting protein kinase 1 (RIPK1) and RIPK2 belong to a family of serine/threonine kinases with conserved domain architecture and important functions in apoptosis, necrosis and innate immunity.ResultsWe found that RIPK1 and RIPK2 but not other members of the RIP kinase family are cleaved by HIV-1 PR. In RIPK1, we identified a putative PR cleavage site; a mutation at this site rendered RIPK1 resistant to PR cleavage. RIPK1 and RIPK2 were cleaved during HIV-1 infection of T cell lines or primary activated CD4+ T cells. Interfering with the viral life cycle at different stages by the addition of specific inhibitors against RT, integrase, or PR, completely prevented RIPK1 and RIPK2 cleavage. Cleavage of RIPK1 disrupted RIPK1/RIPK3 complex formation and RIPK1-mediated induction of NF-kB.ConclusionsThese findings indicate that RIPK1 and RIPK2 are targets of HIV-1 PR activity during infection, and their inactivation may contribute to modulation of cell death and host defense pathways by HIV-1.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0200-6) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2

2015

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“…Infectivity was determined similarly as described for Mason-Pfizer monkey virus [65][66][67][68][69][70][71]. Briefly, culture media from HEK 293 cells transfected with psPAX2, pWPXLd-GFP, and pHEF-VSV-G vectors in a 1:1:1 ratio in the presence of test compounds were collected 48 h post-transfection and filtered through a 0.45-µm filter.…”

Section: Single-round Infectivity Assaymentioning

confidence: 99%

“…The sample preparation and measurements were carried out as described elsewhere [68,70,71]. The VSV-G GFP-HIV infected cells were analyzed with a BD FACS Aria III flow cytometer (Becton Dickinson) with the excitation at 488 nm and the emission separated by a 530/30 band pass filter.…”

Section: Flow Cytometrymentioning

confidence: 99%

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles

et al. 2020

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A major structural retroviral protein, capsid protein (CA), is able to oligomerize into two different hexameric lattices, which makes this protein a key component for both the early and late stages of HIV-1 replication. During the late stage, the CA protein, as part of the Gag polyprotein precursor, facilitates protein–protein interactions that lead to the assembly of immature particles. Following protease activation and Gag polyprotein processing, CA also drives the assembly of the mature viral core. In the early stage of infection, the role of the CA protein is distinct. It controls the disassembly of the mature CA hexameric lattice i.e., uncoating, which is critical for the reverse transcription of the single-stranded RNA genome into double stranded DNA. These properties make CA a very attractive target for small molecule functioning as inhibitors of HIV-1 particle assembly and/or disassembly. Of these, inhibitors containing the PF74 scaffold have been extensively studied. In this study, we reported a series of modifications of the PF74 molecule and its characterization through a combination of biochemical and structural approaches. Our data supported the hypothesis that PF74 stabilizes the mature HIV-1 CA hexameric lattice. We identified derivatives with a higher in vitro stabilization activity in comparison to the original PF74 molecule.

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“…Evaluation of the proportion of dead cells was adapted from Vermes et al [86] MCF-7 cells were plated in a 6-well plate (25,000 cells per well) and treated with compounds 1-4 (0.1-5 µM) for 24 h. Then, the cells were illuminated for 13 min by a 150-W halogen lamp with an edge-pass filter transmitting wavelengths longer than 500 nm (the total light dose of 4 J•cm −2 ). The cells were incubated at standard cultivation conditions for next 24 h. Afterwards, the cells were harvested by trypsinization, washed in cold PBS and resuspended in annexin-binding buffer followed by labeling with Alexa Fluor ® 488 Annexin V and propidium iodide according to the manufacturer's protocol (Dead Cell Apoptosis Kit, Thermo Fisher Scientific, Waltham, MA, USA) as described in Kirakci et al [87] and Rumlová et al [88]. The stained cells were then analyzed by flow cytometer BD FACSAria III, by which live and dead (necrotic and apoptotic) cells were determined using BD FACSDiva 8.…”

Section: Cell Death Evaluation By Flow Cytometrymentioning

confidence: 99%

PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer

et al. 2019

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Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.

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HIV-1 protease-induced apoptosis

Cited by 41 publications

References 62 publications

HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2

HIV-1 protease cleaves the serine-threonine kinases RIPK1 and RIPK2

PF74 and Its Novel Derivatives Stabilize Hexameric Lattice of HIV-1 Mature-Like Particles

PEGylated Purpurin 18 with Improved Solubility: Potent Compounds for Photodynamic Therapy of Cancer

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