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GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity.

ABSTRACT: MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4(+) T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We sought to determine whether GILT's reductase activity regulates CatS expression and function. Confocal microscopy confirmed that GILT and CatS colocalized within lysosomes of B cells. GILT expression posttranscriptionally decreased the steady-state protein expression of CatS in primary B cells and B-cell lines. GILT did not substantially alter the expression of other lysosomal proteins, including H2-M, H2-O, or CatL. GILT's reductase active site was necessary for diminished CatS protein levels, and GILT expression decreased the half-life of CatS, suggesting that GILT-mediated reduction of protein disulfide bonds enhances CatS degradation. GILT expression decreased the proteolysis of a CatS selective substrate. This study illustrates a physiologic mechanism that regulates CatS and has implications for fine tuning MHC class II-restricted Ag processing and for the development of CatS inhibitors, which are under investigation for the treatment of autoimmune disease.

SUBMITTER: Phipps-Yonas H

PROVIDER: S-EPMC3706190 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity.

Phipps-Yonas Hannah H Semik Vikki V Hastings Karen Taraszka KT

European journal of immunology 20121126 1

MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4(+) T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so ...[more]

PMID: 23012103

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Project description:Glycosylated Gag (glycoGag) is an accessory protein expressed by most gammaretroviruses, including murine leukemia virus (MLV). MLV glycoGag not only enhances MLV replication and disease progression but also increases human immunodeficiency virus type 1 (HIV-1) infectivity as Nef does. Recently, SERINC5 (Ser5) was identified as the target for Nef, and the glycoGag Nef-like activity has been attributed to the Ser5 antagonism. Here, we investigated how glycoGag antagonizes Ser5 using MLV glycoMA and murine Ser5 proteins. We confirm previous observations that glycoMA relocalizes Ser5 from plasma membrane to perinuclear punctated compartments and the important role of its Y36XXL39 motif in this process. We find that glycoMA decreases Ser5 expression at steady-state levels and identify two other glycoGag crucial residues, P31 and R63, for the Ser5 downregulation. The glycoMA and Ser5 interaction is detected in live cells using a bimolecular fluorescence complementation assay. Ser5 is internalized via receptor-mediated endocytosis and relocalized to Rab5+ early, Rab7+ late, and Rab11+ recycling endosomes by glycoMA. Although glycoMA is not polyubiquitinated, the Ser5 downregulation requires Ser5 polyubiquitination via the K48- and K63-linkage, resulting in Ser5 destruction in lysosomes. Although P31, Y36, L39, and R63 are not required for glycoMA interaction with Ser5, they are required for Ser5 relocalization to lysosomes for destruction. In addition, although murine Ser1, Ser2, and Ser3 exhibit very poor antiviral activity, they are also targeted by glycoMA for lysosomal destruction. We conclude that glycoGag has a broad activity to downregulate SERINC proteins via the cellular endosome/lysosome pathway, which promotes viral replication.IMPORTANCE MLV glycoGag not only enhances MLV replication but also increases HIV-1 infectivity similarly as Nef. Recent studies have discovered that both glycoGag and Nef antagonize a novel host restriction factor Ser5 and promote viral replication. Compared to Nef, the glycoGag antagonism of Ser5 is still poorly understood. MLV glycoGag is a transmembrane version of the structural Gag protein with an extra 88-amino-acid leader region that determines its activity. We now show that glycoGag interacts with Ser5 in live cells and internalizes Ser5 via receptor-mediated endocytosis. Ser5 is polyubiquitinated and relocalized to endosomes and lysosomes for massive destruction. In addition to the previously identified tyrosine-based sorting signal, we find two more important residues for Ser5 relocalization and downregulation. We also find that the Ser5 sensitivity to glycoGag is conserved in the SERINC family. Together, our findings highlight the important role of endosome/lysosome pathway in the enhancement of viral replication by viral proteins.

Project description:BackgroundThe processes of prostate cancer (PCa) invasion and metastasis are facilitated by proteolytic cascade involving multiple proteases, such as matrix metalloproteinases, serine proteases and cysteine proteases including cathepsin K (CatK). CatK is predominantly secreted by osteoclasts and specifically degrades collagen I leading to bone destruction. PCa and breast cancer preferentially metastasize to the bone. Importantly, CatK expression level is greater in PCa bone metastatic sites compared to primary tumor and normal prostate tissues. However, the underlying mechanism of CatK during PCa metastases into the bone remains to be elucidated. We investigated the functional role of CatK during the PCa establishment and growth process in the murine bone.MethodsCatK mRNA expression was validated by RT-PCR, protein expression by immunoblotting in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Its protein production was measured using ELISA assay. The effect of both knockdowns via siRNA and CatK inhibitor was compared in regard to PCa cell invasion. We further studied the dose-dependent CatK inhibitor effect on conditioned media-induced bone resorption. In setting up an animal model, C4-2B cells were injected into the tibiae of SCID mice. The animals treated with either vehicle or CatK inhibitor for 8 weeks at the time of tumor cell injection (tumor establishment model; protocol I) or 4 weeks after tumor cell injection (tumor progression model; protocol II) were applied to histological and histomorphometric analyses.ResultsWe confirmed CatK expression in PCa LNCaP, C4-2B, and PC3 cells as well as in PCa tissues. Furthermore, we observed the inhibitory effects of a selective CatK inhibitor on PCa cell invasion. The CatK inhibitor dose-dependently inhibited PCa-conditioned media-induced bone resorption. Upon injection of C4-2B cells into the tibiae of SCID mice, the selective CatK inhibitor significantly prevented the tumor establishment in protocol I, and reduced the tumor growth in bone in protocol II. It also decreased serum PSA levels in both animal models. The inhibitory effects of the CatK inhibitor were enhanced in combination with zoledronic acid (ZA).ConclusionThe selective CatK inhibitor may prevent the establishment and progression of PCa in bone, thus making it a novel therapeutic approach for advanced PCa.

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GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity.

Publications

GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity.

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