Calpain is involved in the HIV replication from the latently infected OM10.1 cells

Treatment of OM10.1 cells latently infected with human immunodeficiency virus type 1 (HIV-1) with phorbol ester and calcium ionophore (A23187) induced virus replication which was blocked by N-Ac-Leu-Leu-norleucinal (ALLnL), a calpain inhibitor I, and not by lactacystin, a specific proteasome inhibitor. When the purified NF-kappa B/I kappa B complex was treated with mu-calpain, the specific DNA-binding activity was demonstrated by using electrophoretic mobility shift assay in vitro. This effect of mu-calpain was inhibited by ALLnL and calpastatin and not by lactacystin. In fact, we found that mu-calpain efficiently degraded I kappa B alpha. Furthermore, our Western blotting analysis has revealed that mu-calpain cleaves I kappa B alpha at its N-terminal and C-terminal regions that were previously reported to be involved in the interaction with NF-kappa B p65. These observations indicate that in monocyte/macrophage cells calcium signaling is involved in NF-kappa B activation through activation of calpain and thus calpain inhibitors may be effective in inhibiting the activation of latently infected HIV.     

The potential role of ubiquitin c-terminal hydrolases in oncogenesis

Deubiquitinating enzymes (DUBs), capable of removing ubiquitin (Ub) from protein substrates, are involved in numerous biological processes. The ubiquitin C-terminal hydrolases (UCHs) subfamily of DUBs consists of four members: UCH-L1, UCH-L3, UCH37 and BRCA1-associated protein-1 (BAP1). UCH-L1 possesses deubiquitinating activity and dimerization-dependent ubiquitin ligase activity, and functions as a mono-ubiquitin stabilizer; UCH-L3 does both deubiquitinating and deneddylating activity, except dimerization or ligase activity, and unlike UCH-L1, can interact with Lys48-linked Ub dimers to protect it from degradation and in the meanwhile to inhibit its hydrolase activity; UCH37 is responsible for the deubiquitinating activity in the 19S proteasome regulatory complex, and as indicated by the recent study, UCH37 is also associated with the human Ino80 chromatin-remodeling complex (hINO80) in the nucleus and can be activated via transient association of 19S regulatory particle- or proteasome-bound hRpn13 with hINO80; BAP1, binding to the wild-type BRCA1 RING finger domain, is regarded as a tumor suppressor, but for such suppressing activity, as demonstrated otherwise, both deubiquitinating activity and nucleus localization are required. There is growing evidence that UCH enzymes and human malignancies are closely correlated. Previous studies have shown that UCH enzymes play a crucial role in some signalings and cell-cycle regulation. In this review, we provided an insight into the relation between UCH enzymes and oncogenesis.     

Heat shock protein 27 association with the I kappa B kinase complex regulates tumor necrosis factor alpha-induced NF-kappa B activation

Heat shock protein 27 (Hsp27) is a ubiquitously expressed member of the heat shock protein family that has been implicated in various biological functions including the response to heat shock, oxidative stress, and cytokine treatment. Previous studies have demonstrated that heat shock proteins are involved in regulating signal transduction pathways including the NF-kappa B pathway. In this study, we demonstrated that Hsp27 associates with the I kappa B kinase (IKK) complex and that this interaction was stimulated by tumor necrosis factor alpha treatment. Phosphorylation of Hsp27 by the kinase mitogen-activated protein kinase-activated protein kinase 2, a downstream substrate of the mitogen-activated protein kinase p38, enhanced the association of Hsp27 with IKK beta to result in decreased IKK activity. Consistent with these observations, treatment of cells with a p38 inhibitor reduced the association of Hsp27 with IKK beta and thus resulted in increased IKK activity. These studies indicate that Hsp27 plays a negative role in down-regulating IKK signaling by reducing its activity following tumor necrosis factor alpha stimulation.     

Positional-scanning fluorigenic substrate libraries reveal unexpected specificity determinants of DUBs (deubiquitinating enzymes)

DUBs (deubiquitinating enzymes) are a family of proteases responsible for the specific removal of ubiquitin attached to target proteins and thus control the free cellular pools of this molecule. DUB activity is usually assayed using full-length ubiquitin, and these enzymes generally show low activity towards small substrates that constitute the P4-P1 LRGG (Lys-Arg-Gly-Gly) C-terminal motif of ubiquitin. To gain insight into the C-terminal recognition region of ubiquitin by DUBs, we synthesized positional scanning libraries of fluorigenic tetrapeptides and tested them on three examples of human DUBs [OTU-1 (ovarian tumour 1), Iso-T (isopeptidase T) and UCH-L3 (ubiquitin C-terminal hydrolase L3)] and one viral ubiquitin-specific protease, namely PLpro (papain-like protease) from SARS (severe acute respiratory syndrome) virus. In most cases the results show flexibility in the P4 position, very high specificity for arginine in the P3 position and glycine in the P2 position, in accord with the sequence of the natural substrate, ubiquitin. Surprisingly, screening of the P2 position revealed that UCH-L3, in contrast with all the other tested DUBs, demonstrates substantial tolerance of alanine and valine at P2, and a parallel analysis using the appropriate mutation of the full-length ubiquitin confirms this. We have also used an optimal tetrapeptide substrate, acetyl-Lys-Arg-Gly-Gly-7-amino-4-methylcoumarin, to investigate the activation mechanism of DUBs by ubiquitin and elevated salt concentration. Together, our results reveal the importance of the dual features of (1) substrate specificity and (2) the mechanism of ubiquitin binding in determining deubiquitination by this group of proteases.     

Failure of measles virus to activate nuclear factor-kappa B in neuronal cells: implications on the immune response to viral infections in the central nervous system

Neurons are postmitotic cells that foster virus persistence. These cells lack the HLA class I molecules required for clearance of infected cells. Previously, we showed that HLA class I is induced by measles virus (MV) on glial cells, which is primarily mediated by IFN-beta. In contrast, MV was unable to induce HLA class I or IFN-beta in neuronal cells. This failure was associated with lack of NF-kappa B binding to the positive regulatory domain II element of the IFN-beta promoter, which is essential for virus-induced IFN-beta gene activity. In this study, we demonstrate that the failure to activate NF-kappa B in neuronal cells is due to the inability of MV to induce phosphorylation and degradation of I kappa B, the inhibitor of NF-kappa B. In contrast, TNF-alpha induced degradation of I kappa B alpha in the neuronal cells, suggesting that failure to induce I kappa B alpha degradation is likely due to a defect in virus-mediated signaling rather than to a defect involving neuronal I kappa B alpha. Like MV, mumps virus and dsRNA failed to induce I kappa B alpha degradation in the neuronal cells, suggesting that this defect may be specific to viruses. Autophosphorylation of the dsRNA-dependent protein kinase, a kinase possibly involved in virus-mediated I kappa B alpha phosphorylation, was intact in both cell types. The failure of virus to induce I kappa B alpha phosphorylation and consequently to activate NF-kappa B in neuronal cells could explain the repression of IFN-beta and class I gene expression in virus-infected cells. These findings provide a potential mechanism for the ability of virus to persist in neurons and to escape immune surveillance.