Autoantibodies from primary biliary cirrhosis patients with anti-p95c antibodies bind to recombinant p97/VCP and inhibitin vitronuclear envelope assembly

Abstract

SUMMARYWe have reported previously that p95c, a novel 95-kDa cytosolic protein, was the target of autoantibodies in sera of patients with autoimmune hepatic diseases. We studied 30 sera that were shown previously to immunoprecipitate a 95 kDa protein from [35 S]-methionine-labelled HeLa lysates and had a specific precipitin band in immunodiffusion. Thirteen sera were available to test the ability of p95c antibodies to inhibit nuclear envelope assembly in an in vitro assay in which confocal fluorescence microscopy was also used to identify the stages at which nuclear assembly was inhibited. The percentage inhibition of nuclear envelope assembly of the 13 sera ranged from 7% to 99% and nuclear envelope assembly and the swelling of nucleus was inhibited at several stages. The percentage inhibition of nuclear assembly was correlated with the titre of anti-p95c as determined by immunodiffusion. To confirm the identity of this autoantigen, we used a full-length cDNA of the p97/valosin-containing protein (VCP) to produce a radiolabelled recombinant protein that was then used in an immunoprecipitation (IP) assay. Our study demonstrated that 12 of the 13 (93%) human sera with antibodies to p95c immunoprecipitated recombinant p97/VCP. Because p95c and p97 have similar molecular masses and cell localization, and because the majority of sera bind recombinant p97/VCP and anti-p95c antibodies inhibit nuclear assembly, this is compelling evidence that p95c and p97/VCP are identical.

Sorafenib-Mediated Targeting of the AAA+ ATPase p97/VCP Leads to Disruption of the Secretory Pathway, Endoplasmic Reticulum Stress, and Hepatocellular Cancer Cell Death

“…Proteomics studies have suggested that p97/VCP might play some role in the liver regeneration and in fetal liver development (31), and it has also been shown that p97/VCP contributes to the formation of Mallory bodies (32). Moreover, p97/VCP autoantibodies have been found in cases of autoimmune hepatitis (33), primary biliary cirrhosis (34,35), and in HCC (36). Elevated expression of p97/VCP in HCC has also been associated with increased incidence of tumor recurrence (37).…”

Nuclear localization of valosin‐containing protein in normal muscle and muscle affected by inclusion‐body myositis

“…[3][4][5]8 The localization of VCP to myonuclei shown here suggests that some muscle pathology in IBMPFD may result from a disturbance of the nuclear function of VCP, which includes the maintenance and assembly of nuclear membrane. 10,15 In this regard, nuclear pathology consisting of ubiquitinated nuclear inclusions 6 and TAR DNA binding protein 43 (TDP-43) nuclear inclusions 16 have been described in IBMPFD neurons, and a hypothesis has been suggested that …”

“…18 VCP has a range of reported functions, including the assembly of nuclear membranes, 10,15 but the specific mechanism that results in tissue pathology in IBMPFD is unknown. The location of VCP has been studied in several mammalian cell culture lines, where it is present in the cytoplasm and nucleus.…”

Probing the mRNA processing body using protein macroarrays and "autoantigenomics"

“…These proteins included four known P-body components, DCP1a, TTP, Ge-1, and RAP55 (van Dijk et al 2002;Fenger-Gron et al 2005;Yu et al 2005;Anderson and Kedersha 2006;Yang et al 2006). In addition to Ge-1 and RAP55, three other PBC autoantigens, E3 pyruvate dehydrogenase complex, lamin A/C, and valosin-containing protein, were identified (Courvalin and Worman 1997;Palmer et al 1999;Miyachi et al 2004). Some of the sera reacted with autoantigens that are not usually associated with PBC, including CALR, GRIPAP1, SART1, and U1A (Bringmann et al 1983;McCauliffe et al 1990;Shichijo et al 1998;Stinton et al 2005).…”