Inhibition of Infectious HIV-1 Production by Rerouting the Cellular Furin Inhibitor Serpin B8.

Serpins are a superfamily of proteins that regulate a variety of physiological processes by irreversibly inhibiting the enzymatic activity of different serine proteases. For example, Serpin Family B Member 8 (Serpin B8, also known as PI8 and CAP2) binds to and inhibits the proprotein convertase furin. Like many other viral pathogens, human immunodeficiency virus type 1 (HIV-1) exploits furin for the proteolytic activation of its envelope glycoprotein (Env). Since the furin inhibitor Serpin B8 is expressed in primary target cells of HIV-1 and induced under inflammatory conditions, we hypothesized that it might interfere with HIV-1 Env maturation and decrease infectivity of newly produced virions. Indeed, recombinant Serpin B8 reduced furin-mediated cleavage of an HIV-1 Env reporter substrate in vitro. However, Serpin B8 did not affect Env maturation or reduce HIV-1 particle infectivity when expressed in HIV-1-producing cells. Immunofluorescence imaging, dimerization assays and in silico sequence analyses revealed that Serpin B8 failed to inhibit intracellular furin since both proteins localized to different subcellular compartments. We therefore aimed at rendering Serpin B8 active against HIV-1 by relocalizing it to furin-containing secretory compartments. Indeed, the addition of a heterologous signal peptide conferred potent anti-HIV-1 activity to Serpin B8 and significantly decreased infectivity of newly produced viral particles. Thus, our findings demonstrate that subcellular relocalization of a cellular protease inhibitor can result in efficient inhibition of infectious HIV-1 production. IMPORTANCE Many cellular proteases serve as dependency factors during viral infection and are hijacked by viruses for the maturation of their own (glyco)proteins. Consequently, inhibition of these cellular proteases may represent a means to inhibit the spread of viral infection. For example, several studies have investigated the serine protease furin as a potential therapeutic target since this protease cleaves and activates several viral envelope proteins, including HIV-1 Env. Besides the development of small molecule inhibitors, cell-intrinsic protease inhibitors may also be exploited to advance current antiviral treatment approaches. Here, we show that Serpin B8, an endogenous furin inhibitor, can inhibit HIV-1 Env maturation and efficiently reduce infectious HIV-1 production when rerouted to the secretory pathway. The results of our study not only provide important insights into the biology of Serpins, but also show how protein engineering of an endogenous furin inhibitor can render it active against HIV-1.

Mitofusins modulate the increase in mitochondrial length, bioenergetics and secretory phenotype in therapy-induced senescent melanoma cells.

Cellular senescence is an endpoint of chemotherapy, and targeted therapies in melanoma and the senescence-associated secretory phenotype (SASP) can affect tumor growth and microenvironment, influencing treatment outcomes. Metabolic interventions can modulate the SASP, and an enhanced mitochondrial energy metabolism supports resistance to therapy in melanoma cells. Herein, we assessed the mitochondrial function of therapy-induced senescent melanoma cells obtained after exposing the cells to temozolomide (TMZ), a methylating chemotherapeutic agent. Senescence induction in melanoma was accompanied by a substantial increase in mitochondrial basal, ATP-linked, and maximum respiration rates and in coupling efficiency, spare respiratory capacity, and respiratory control ratio. Further examinations revealed an increase in mitochondrial mass and length. Alterations in mitochondrial function and morphology were confirmed in isolated senescent cells, obtained by cell-size sorting. An increase in mitofusin 1 and 2 (MFN1 and 2) expression and levels was observed in senescent cells, pointing to alterations in mitochondrial fusion. Silencing mitofusin expression with short hairpin RNA (shRNA) prevented the increase in mitochondrial length, oxygen consumption rate and secretion of interleukin 6 (IL-6), a component of the SASP, in melanoma senescent cells. Our results represent the first in-depth study of mitochondrial function in therapy-induced senescence in melanoma. They indicate that senescence increases mitochondrial mass, length and energy metabolism; and highlight mitochondria as potential pharmacological targets to modulate senescence and the SASP.

Targeting TMEM176B Enhances Antitumor Immunity and Augments the Efficacy of Immune Checkpoint Blockers by Unleashing Inflammasome Activation.

Although immune checkpoint blockers have yielded significant clinical benefits in patients with different malignancies, the efficacy of these therapies is still limited. Here, we show that disruption of transmembrane protein 176B (TMEM176B) contributes to CD8+ T cell-mediated tumor growth inhibition by unleashing inflammasome activation. Lack of Tmem176b enhances the antitumor activity of anti-CTLA-4 antibodies through mechanisms involving caspase-1/IL-1ß activation. Accordingly, patients responding to checkpoint blockade therapies display an activated inflammasome signature. Finally, we identify BayK8644 as a potent TMEM176B inhibitor that promotes CD8+ T cell-mediated tumor control and reinforces the antitumor activity of both anti-CTLA-4 and anti-PD-1 antibodies. Thus, pharmacologic de-repression of the inflammasome by targeting TMEM176B may enhance the therapeutic efficacy of immune checkpoint blockers.

Polypeptide-GalNAc-T6 expression predicts better overall survival in patients with colon cancer.

Colorectal carcinoma (CRC) is the second leading cause of cancer mortality worldwide. O-glycosylated mucins at the cell surface of colonic mucosa exhibit alterations in cancer and are involved in fundamental biological processes, including invasion and metastasis. Certain members of the GalNAc-transferase family may be responsible for these changes and are being investigated as novel biomarkers of cancer. In the present study the prognostic significance of GalNAc-T6 was investigated in patients with CRC patients. GalNAc-T6 expression was observed in all three colon cancer cell lines analyzed by reverse transcription-polymerase chain reaction, immunofluorescence and flow cytometry. A cohort of 81 colon cancer specimens was analyzed by immunohistochemical staining using MAb T6.3. It was demonstrated that GalNAc-T6 was expressed in 35/81 (43%) cases of colon cancer but not in the normal colonic mucosa. No association was observed with the clinical-pathologic parameters. However, patients expressing GalNAc-T6 had a significantly increased overall survival (median, 58 months; P<0.001) compared with GalNAc-T6 negative patients, especially those with advanced disease. These results suggest that GalNAc-T6 expression predicts an improved outcome in patients with CRC. The molecular mechanism underlying the less aggressive behavior of colon cancer cells expressing GalNAc-T6 remains to be elucidated.

A green multicomponent synthesis of tocopherol analogues with antiproliferative activities.

A one-pot efficient, practical and eco-friendly synthesis of tocopherol analogues has been developed using water or solvent free conditions via Passerini and Ugi multicomponent reactions. These reactions can be optimized using microwave irradiation or ultrasound as the energy source. Accordingly, a small library of 30 compounds was prepared for biological tests. The evaluation of the antiproliferative activity in the human solid tumor cell lines A549 (lung), HBL-100 (breast), HeLa (cervix), SW1573 (lung), T-47D (breast), and WiDr (colon) provided lead compounds with GI50 values between 1 and 5 µM. A structure-activity relationship is also discussed. One of the studied compounds comes up as a future candidate for the development of potent tocopherol-mimetic therapeutic agents for cancer.

Data for comparative proteomics analysis of the antitumor effect of CIGB-552 peptide in HT-29 colon adenocarcinoma cells.

CIGB-552 is a second generation antitumor peptide that displays potent cytotoxicity in lung and colon cancer cells. The nuclear subproteome of HT-29 colon adenocarcinoma cells treated with CIGB-552 peptide was identified and analyzed [1]. This data article provides supporting evidence for the above analysis.

Comparative proteomics analysis of the antitumor effect of CIGB-552 peptide in HT-29 colon adenocarcinoma cells.

The second generation peptide CIGB-552 has a pro-apoptotic effect on H460 non-small cell lung cancer cells and displays a potent cytotoxic effect in HT-29 colon adenocarcinoma cells though its action mechanism is ill defined. Here, we present the first proteomic study of peptide effect in HT-29 cells using subcellular fractionation, protein and peptide fractionation by DF-PAGE and LC-MS/MS peptide identification. In particular, we explored the nuclear proteome of HT-29 cells at a 5h treatment identifying a total of 68 differentially modulated proteins, 49 of which localize to the nucleus. The differentially modulated proteins were analyzed following a system biology approach. Results pointed to a modulation of apoptosis, oxidative damage removal, NF-κB activation, inflammatory signaling and of cell adhesion and motility. Further Western blot and flow-cytometry experiments confirmed both pro-apoptotic and anti-inflammatory effects of CIGB-552 peptide in HT-29 cells.

Activation of Toll-like receptor 2 increases macrophage resistance to HIV-1 infection.

Patients infected with HIV-1, the etiological agent of AIDS, have increased intestinal permeability, which allows for the passage of microbial products, including Toll-like receptor (TLR) ligands, into circulation. The exposure of HIV-1-infected cells to certain TLR agonists affects viral replication, but studies associating viral production with the activation of TLR2 in HIV-1-infected cells are rare and controversial. Here, we report that the TLR2 ligands Zymosan and Pam3CSK4 potently inhibit HIV-1 replication in acutely infected monocyte-derived macrophages and the exposure to TLR2 ligands prior to infection renders macrophages refractory to HIV-1 production. Macrophage treatment with Pam3CSK4 did not change the cellular expression of the HIV-1 entry receptors CD4 and CCR5. Both TLR2 ligands increased the macrophage production of ß-chemokines and IL-10, and the blockage of these soluble factors prevented the inhibitory effect of TLR2 activation on HIV-1 replication. Our findings show that the direct engagement of TLR2 in HIV-1-infected macrophages increase cellular resistance to HIV-1 infection, and that controlling HIV-1 replication with agonists for TLR2 might have implications for the development of antiretroviral therapies.