Autoantibodies to citrullinated protein antigens are specific markers of rheumatoid arthritis (RA). Although protein citrullination can be activated by numerous stimuli in cells, it remains unclear which of these produce the prominent citrullinated autoantigens targeted in RA. In these studies, we show that RA synovial fluid cells have an unusual pattern of citrullination with marked citrullination of proteins across the broad range of molecular weights, which we term cellular hypercitrullination. Although histone citrullination is a common event during neutrophil activation and death induced by different pathways including apoptosis, NETosis, and necroptosis/autophagy, hypercitrullination is not induced by these stimuli. However, marked hypercitrullination is induced by two immune-mediated membranolytic pathways, mediated by perforin and the membrane attack complex (MAC), which are active in the RA joint and of importance in RA pathogenesis. We further demonstrate that perforin and MAC activity on neutrophils generate the profile of citrullinated autoantigens characteristic of RA. These data suggest that activation of peptidylarginine deiminases during complement and perforin activity may be at the core of citrullinated autoantigen production in RA. These pathways may be amenable to monitoring and therapeutic modulation.

Granzymes (granule enzymes) are proteases released from cytotoxic lymphocyte granules into target cells to protect mammals from virus infection and transformed cells. Once released into the cytoplasm of the target cell, granzymes activate specific pathways to induce cell death. Although the induction of target cell death has been considered the central function for these proteases, accumulating evidence suggests that granzymes also possess additional non-death-related functions. Thus, some granzymes can achieve direct antiviral activities through the cleavage of proteins encoded by viruses as well as host factors required for the viral life cycle. The presence of elevated concentrations of circulating granzymes in various inflammatory processes and granzyme-mediated cleavage of extracellular substrates suggest that these proteases may have extracellular effects relevant to virus and tumor rejection and the pathogenesis of chronic inflammatory diseases. Here, we discuss the current knowledge of the substrates and the proposed non-apoptotic functions of granzymes, with special interest in non-death-related functions of granzymes inside the target cell.

Granzymes are key components of the cytotoxic arm of the immune response, which play critical roles in eliminating host cells infected by intracellular pathogens and transformed cells. Although the induction of cell death is likely a central process underlying the function of these enzymes, little is known about whether granzymes use additional mechanisms to exert their antipathogen activity. This study identifies La, a phosphoprotein involved in multiple roles in cellular and viral RNA metabolism, as the first nonapoptotic substrate of granzyme H (gzmH), a cytotoxic granule protease that is constitutively expressed by NK cells. Cleavage of La by gzmH occurs at Phe-364 (P 1 site) and generates a COOH-terminal truncated form of La that loses nuclear localization and decreases HCV (hepatitis C virus)-internal ribosome entry site (IRES)-mediated translational activity. The ability of gzmH to cleave host proteins involved in essential viral functions provides a novel mechanism by which granzymes can mediate direct antiviral activities.

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Objective To address the independent roles of peptidylarginine deiminase type 2 (PAD2) and PAD4 in generating rheumatoid arthritis (RA) autoantigens by using a system that mimics intracellular citrullination in the RA joint. Methods PAD2‐ or PAD4‐expressing 293T cells and mock‐transfected cells were used as targets in cytotoxic assays using lymphokine‐activated killer cells, cytotoxic YT cell granule contents, or purified human perforin. Protein citrullination and autoantigen production were determined by immunoblotting using the anti–modified citrulline–Senshu method and RA sera (n = 30), respectively. Results RA sera recognized at least 3 categories of autoantigens in PAD‐expressing target cells killed by the cytotoxic lymphocyte granule–induced death pathway. These included: 1) autoantigens targeted in their native form, 2) citrullinated antigens, and 3) antigens cleaved by cytotoxic proteases (e.g., granzymes). Interestingly, although target cells expressing PAD2 or PAD4 showed prominent hypercitrullination of a broad range of proteins during cytotoxic granule–induced cell damage, autoantibodies in RA sera targeted only a very limited number of antigens in hypercitrullinated cells. Furthermore, RA sera showed distinct reactivities to autoantigens generated by PAD2 or PAD4. Conclusion The cytotoxic granule–induced death pathway has the capacity to modify antigens by inducing hypercitrullination and antigen cleavage in target cells. Interestingly, among a large number of citrullinated proteins generated by PAD2 and PAD4 in cells, only a few are likely involved in the production of autoantibodies in RA.

Gpn3 is required for RNA polymerase II (RNAPII) nuclear targeting. Here, we investigated the effect of a cancer-associated Q279* nonsense mutation in Gpn3 cellular function. Employing RNAi, we replaced endogenous Gpn3 by wt or Q279* RNAi-resistant Gpn3R in epithelial model cells. RNAPII nuclear accumulation and transcriptional activity were markedly decreased in cells expressing only Gpn3R Q279*. Wild-type Gpn3R localized to the cytoplasm but a fraction of Gpn3R Q279* entered the cell nucleus and inhibited Gpn1-EYFP nuclear export. This property and the transcriptional deficit in Gpn3R Q279*-expressing cells required a PDZ-binding motif generated by the Q279* mutation. We conclude that an acquired PDZ-binding motif in Gpn3 Q279* caused Gpn3 nuclear entry, and inhibited Gpn1 nuclear export and Gpn3-mediated RNAPII nuclear targeting.

Background Perforin- and complement-induced cell lysis has recently been implicated in the generation of citrullinated autoantigens in rheumatoid arthritis (RA). By activating PADs in target cells, immune-mediated pore-forming pathways induce abnormal protein citrullination in the RA joint. Here, we demonstrate a novel approach to identify unique autoantibody reactivities to citrullinated proteins generated by the perforin pathway. Objectives To identify novel perforin-induced autoantigens in RA, and to characterize the antibody response to citrullinated hnRNP A1 (cit-hnRNP A1) in patients with RA. Methods 293T cells transiently transfected to express human PAD2 or PAD4 were incubated with perforin from cytotoxic granule contents (GC) to induce PAD activation. Cellular citrullination was confirmed by anti-modified citrulline (AMC) immunoblotting. Inducible autoantigens generated during perforin-mediated cytolysis were identified by immunoblotting using RA patient sera. Antigen identity was determined by 2D gel electrophoresis/mass spectrometry (MS). Recombinant hnRNP A1 was expressed in E. coli. Patients with RA (n=196) from a longitudinal cohort study (ESCAPE RA) and healthy controls (n=56) were assayed for IgG antibodies to unmodified and in vitro citrullinated hnRNP A1 by ELISA. Positivity was defined as an arbitrary unit (AU) above the 98th percentile of controls. Anti-cit-hnRNP A1 antibody specificity was determined by subtracting reactivities measured against unmodified hnRNP A1 from reactivities against cit-hnRNP A1. Results 293T cells expressing PAD2 or PAD4 were used to study PAD-isotype specific autoantigen citrullination. A 34-kDa autoantigen inducible in both PAD2- and PAD4-transfected cells was distinctly targeted by RA patient sera (Fig. 1A; GC: perforin-treated cells, NT: untreated controls). MS identified the modified protein as cit-hnRNP A1, and citrullination by both rPAD2 and rPAD4 was confirmed using purified hnRNP A1. Antibody reactivity against cit-hnRNP A1 was detected in 70 (36%) of RA patients vs. only 1 (2%) of controls (p<0.0001: Figure 1B; HC: healthy controls), and anti-cit-hnRNP A1 specific antibodies were confirmed in 65 (33%) of RA patients. In contrast, the low-titer reactivities against unmodified hnRNP A1 observed in patients with RA did not differ significantly from controls (7% vs. 2%; p=0.32) (Fig. 1B). Anti-cit-hnRNP A1 positivity in RA was associated with markers of erosive disease as measured using the Sharp-van der Heijde score (median 12 vs. 6 units, respectively for antibody positive vs. negative; p=0.019) and total erosion score (median 6 vs. 2 units, respectively; p=0.008) despite equivalent therapeutic intervention. Interestingly, this marker uniquely associated with elevated levels of IL-6 (58% higher in antibody positive vs. negative; p=0.01). Figure 1 Conclusions These studies identify hnRNP A1 as a citrullinated autoantigen in RA generated during perforin-mediated cytotoxicity. Anti-cit-hnRNP A1 antibodies may serve as a novel biomarker associated wit...