Naveen Bangia

Role for proteasome activator PA200 and postglutamyl proteasome activity in genomic stability

Proteasome activator PA200 enhances proteasome-mediated cleavage after acidic residues in vitro; however, its role within cells is not known. Here, we show that, in response to ionizing radiation, PA200 forms hybrid proteasomes with 19S caps and 20S core proteasomes that accumulate on chromatin, leading to an increase in proteolytic activity. Unlike many other proteins that respond to DNA damage, the response of PA200 appears to be independent of Ataxia Telangiectasia Mutated and p53, but dependent on DNA-dependent protein kinase activity. Nonetheless, PA200 is critical because PA200-knockdown cells show genomic instability and reduced survival after exposure to ionizing radiation. This phenotype is reproduced by specific inhibition of postglutamyl activity of proteasomes, but combined treatment with PA200 siRNA and postglutamyl inhibitor does not show additive effects on survival. Together, these data suggest a unique role for PA200 in genomic stability that is likely mediated through its ability to enhance postglutamyl cleavage by proteasomes.

Biological effects and clinical significance of lenalidomide-induced tumour flare reaction in patients with chronic lymphocytic leukaemia: in vivo evidence of immune activation and antitumour response.

Lenalidomide has demonstrated impressive antileukaemic effects in patients with chronic lymphocytic leukaemia (CLL). The mechanism(s) by which it mediates these effects remain unclear. Clinically, CLL patients treated with lenalidomide demonstrate an acute inflammatory reaction, the tumour flare reaction that is suggestive of an immune activation phenomenon. Samples from CLL patients treated with lenalidomide were used to evaluate its effect on the tumour cell and components of its microenvironment (immune cellular and cytokine). Lenalidomide was unable to directly induce apoptosis in CLL cells in vitro, however it modulated costimulatory (CD80, CD83, CD86) surface molecules on CLL cells in vitro and in vivo. Concurrently, we demonstrated that NK cell proliferation was induced by lenalidomide treatment in patients and correlated with clinical response. Cytokine analysis showed increase in levels of TNF‐α post‐lenalidomide treatment, consistent with acute inflammatory reaction. Furthermore, the basal cytokine profile (high IL‐8, MIG, IP‐10 and IL‐4 levels and low IL‐5, MIP1a, MIP1b, IL12/p70) was predictive of clinical response to lenalidomide. Collectively, our correlative studies provide further evidence that the antileukaemic effect of lenalidomide in CLL is mediated not only through modulation of the leukaemic clone but also through elements of the tumour microenvironment.

Proteasomes and Proteasome Activator 200 kDa (PA200) Accumulate on Chromatin in Response to Ionizing Radiation

Abstract Blickwedehl, J., McEvoy, S., Wong, I., Kousis, P., Clements, J., Elliott, R., Cresswell, P. Liang, P. and Bangia, N. Proteasomes and Proteasome Activator 200 kDa (PA200) Accumulate on Chromatin in Response to Ionizing Radiation. Radiat. Res. 167, 663–674 (2007). Proteasome activator 200 kDa (PA200) forms nuclear foci after exposure of cells to ionizing radiation and enhances proteasome activity in vitro. Within cells, it is unclear whether PA200 responds to radiation alone or in association with proteasomes. In the present study, we identified three forms of cellular PA200 (termed PA200i, ii and iii) at the mRNA and protein levels. Neither PA200ii nor PA200iii appears to associate with proteasomes. All detectable PA200i is associated with proteasomes, which indicates that PA200i and proteasomes function together within the cell. Consistent with this idea, we find that exposure of cells to radiation leads to an equivalent accumulation of both PA200i and core proteasomes on chromatin. This increase in PA200 and proteasomes on chromatin is not specific to the stage of cell cycle arrest since it occurs in cells that arrest in G2/M and cells that arrest in G1/S after exposure to radiation. These data provide evidence that PA200 and proteasomes function together within cells and respond to a specific radiation-induced damage independent of the stage of cell cycle arrest.

Regulation of CD20 in Rituximab-Resistant Cell Lines and B-cell Non-Hodgkin Lymphoma

Purpose: The aim of this research was to further investigate the contribution of CD20 antigen expression to rituximab activity and define the mechanisms responsible for CD20 downregulation in rituximab-resistant cell lines (RRCL). Experimental Design: Rituximab-sensitive cell lines, RRCL, and primary neoplastic B cells were evaluated by chromium-51 release assays, ImageStream image analysis, immunohistochemical staining, flow cytometric analysis, CD20 knockdown, promoter activity, chromatin immunoprecipitation (ChIP) analysis of CD20 promoter, and CD20 plasmid transfection experiments to identify mechanisms associated with CD20 regulation in RRCL. Results: RRCL exhibited a gradual loss of CD20 surface expression with repeated exposure to rituximab. We identified a CD20 antigen surface threshold level required for effective rituximab-associated complement-mediated cytotoxicity (CMC). However, a direct correlation between CD20 surface expression and rituximab-CMC was observed only in rituximab-sensitive cell lines. CD20 promoter activity was decreased in RRCL. Detailed analysis of various CD20 promoter fragments suggested a lack of positive regulatory factors in RRCL. ChIP analysis showed reduced binding of several key positive regulatory proteins on CD20 promoter in RRCL. Interleukin-4 (IL-4) induced higher CD20 promoter activity and CD20 expression but modestly improved rituximab activity in RRCL and in primary B-cell lymphoma cells. Forced CD20 expression restored cytoplasmic but not surface CD20, suggesting the existence of a defect in CD20 protein transport in RRCL. Conclusions: We identified several mechanisms that alter CD20 expression in RRCL and showed that, whereas CD20 expression is important for rituximab activity, additional factors likely contribute to rituximab sensitivity in B-cell lymphoma. Clin Cancer Res; 18(4); 1039–50. ©2012 AACR.

Stoichiometric tapasin interactions in the catalysis of major histocompatibility complex class I molecule assembly

The assembly of major histocompatibility complex (MHC) class I molecules with their peptide ligands in the endoplasmic reticulum (ER) requires the assistance of many proteins that form a multimolecular assemblage termed the ‘peptide‐loading complex’. Tapasin is the central stabilizer of this complex, which also includes the transporter associated with antigen processing (TAP), MHC class I molecules, the ER chaperone, calreticulin, and the thiol‐oxidoreductase ERp57. In the present report, we investigated the requirements of these interactions for tapasin protein stability and MHC class I dissociation from the peptide‐loading complex. We established that tapasin is stable in the absence of either TAP or MHC class I interaction. In the absence of TAP, tapasin interaction with MHC class I molecules is long‐lived and results in the sequestration of existing tapasin molecules. In contrast, in TAP‐sufficient cells, tapasin is re‐utilized to interact with and facilitate the assembly of many MHC class I molecules sequentially. Furthermore, chemical cross‐linking has been utilized to characterize the interactions within this complex. We demonstrate that tapasin and MHC class I molecules exist in a 1 : 1 complex without evidence of higher‐order tapasin multimers. Together these studies shed light on the tapasin protein life cycle and how it functions in MHC class I assembly with peptide for presentation to CD8+ T cells.

Pseudomonas aeruginosa Cif Protein Enhances the Ubiquitination and Proteasomal Degradation of the Transporter Associated with Antigen Processing (TAP) and Reduces Major Histocompatibility Complex (MHC) Class I Antigen Presentation

Background: P. aeruginosa Cif degrades the ABC transporters CFTR and P-glycoprotein. Results: Cif increases the ubiquitination and degradation of TAP1 and decreases MHC class I antigen presentation in airway epithelial cells. Conclusion: Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation. Significance: These observations suggest a mechanism whereby Pseudomonas infection increases the severity and duration of respiratory viral infections. Cif (PA2934), a bacterial virulence factor secreted in outer membrane vesicles by Pseudomonas aeruginosa, increases the ubiquitination and lysosomal degradation of some, but not all, plasma membrane ATP-binding cassette transporters (ABC), including the cystic fibrosis transmembrane conductance regulator and P-glycoprotein. The goal of this study was to determine whether Cif enhances the ubiquitination and degradation of the transporter associated with antigen processing (TAP1 and TAP2), members of the ABC transporter family that play an essential role in antigen presentation and intracellular pathogen clearance. Cif selectively increased the amount of ubiquitinated TAP1 and increased its degradation in the proteasome of human airway epithelial cells. This effect of Cif was mediated by reducing USP10 deubiquitinating activity, resulting in increased polyubiquitination and proteasomal degradation of TAP1. The reduction in TAP1 abundance decreased peptide antigen translocation into the endoplasmic reticulum, an effect that resulted in reduced antigen available to MHC class I molecules for presentation at the plasma membrane of airway epithelial cells and recognition by CD8+ T cells. Cif is the first bacterial factor identified that inhibits TAP function and MHC class I antigen presentation.

Differential contribution of TAP and tapasin to HLA class I antigen expression

Expression of class I human leucocyte antigens (HLA) on the surface of malignant cells is critical for their recognition and destruction by cytotoxic T lymphocytes. Surface expression requires assembly and folding of HLA class I molecules in the endoplasmic reticulum with the assistance of proteins such as Transporter associated with Antigen Processing (TAP) and tapasin. Interferon‐γ induces both TAP and tapasin so dissection of which protein contributes more to HLA class I expression has not been possible previously. In this study, we take advantage of a human melanoma cell line in which TAP can be induced, but tapasin cannot. Interferon‐γ increases TAP protein levels dramatically but HLA class I expression at the cell surface does not increase substantially, indicating that a large increase in peptide supply is not sufficient to increase HLA class I expression. On the other hand, transfection of either allelic form of tapasin (R240 or T240) enhances HLA‐B*5001 and HLA‐B*5701 antigen expression considerably with only a modest increase in TAP. Together, these data indicate that in the presence of minimal TAP activity, tapasin can promote substantial HLA class I expression at the cell surface.

WHAT IS THE ROLE OF ALTERNATE SPLICING IN ANTIGEN PRESENTATION BY MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I MOLECULES

The expression of major histocompatibility complex (MHC) class I molecules on the cell surface is critical for recognition by cytotoxic T lymphocytes (CTL). This recognition event leads to destruction of cells displaying MHC class I—viral peptide complexes or cells displaying MHC class I—mutant peptide complexes. Before they can be transported to the cell surface, MHC class I molecules must associate with their peptide ligand in the endoplasmic reticulum (ER) of the cell. Within the ER, numerous proteins assist in the appropriate assembly and folding of MHC class I molecules. These include the heterodimeric transporter associated with antigen processing (TAP1 and TAP2), the heterodimeric chaperone-oxidoreductase complex of tapasin and ERp57 and the general ER chaperones calreticulin and calnexin. Each of these accessory proteins has a well-defined role in antigen presentation by MHC class I molecules. However, alternate splice forms of MHC class I heavy chains, TAP and tapasin, have been reported suggesting additional complexity to the picture of antigen presentation. Here, we review the importance of these different accessory proteins and the progress in our understanding of alternate splicing in antigen presentation.

Antigen presentation machinery (APM) modulation and soluble HLA molecules in the tumor microenvironment: do they provide tumor cells with escape mechanisms from recognition by cytotoxic T lymphocytes?

The crucial role played by HLA class I antigens in the interactions of malignant cells with HLA class I antigen restricted, tumor antigen-specific cytotoxic T lymphocytes (CTL) and with NK cells ha...

Aggregate Formation by ERp57-Deficient MHC Class I Peptide-Loading Complexes

The endoplasmic reticulum (ER)‐resident proteins TAP, tapasin and ERp57 are the core components of the major histocompatibility complex (MHC) class I peptide‐loading complex and play an important role in peptide loading by MHC class I‐β2microglobulin dimers. ERp57 and tapasin form a stable disulfide‐linked heterodimer within the peptide‐loading complex. We demonstrate that ERp57‐deficient loading complexes, obtained by expression in a tapasin‐negative cell line of a tapasin mutant (C95A) that is not able to form a disulfide bond with ERp57, are prone to aggregation. We studied the assembly, stability and aggregation of the core loading complex using cell lines stably expressing fluorescently tagged tapasin (wild type or C95A mutant) and TAP1. Part of the loading complexes containing the tagged C95A tapasin and TAP1 were sequestered in the ER, without change of their ER transmembrane topology, and were surrounded by a mesh of filaments at the cytosolic side, resulting in formation of protein aggregates with characteristic morphology. Protein aggregates were associated with changes in ER protein turnover but did not affect the cell viability and did not induce the unfolded protein response. Fluorescence resonance energy transfer analysis of the aggregate‐free ER fraction revealed that lack of ERp57 did not affect the stoichiometry or stability of tapasin–TAP1 interactions in the assembled ‘soluble’ core loading complexes. We conclude that the presence of ERp57 is important for the stability of core loading complexes, and that in its absence, the core loading complexes may form stable aggregates within the ER.