A novel conserved isoform of the ubiquitin ligase UFD2a/UBE4B is expressed exclusively in mature striated muscle cells.

Yeast Ufd2p was the first identified E4 multiubiquitin chain assembly factor. Its vertebrate homologues later referred to as UFD2a, UBE4B or E4B were also shown to have E3 ubiquitin ligase activity. UFD2a function in the brain has been well established in vivo, and in vitro studies have shown that its activity is essential for proper condensation and segregation of chromosomes during mitosis. Here we show that 2 alternative splice forms of UFD2a, UFD2a-7 and -7/7a, are expressed sequentially during myoblast differentiation of C2C12 cell cultures and during cardiotoxin-induced regeneration of skeletal muscle in mice. UFD2a-7 contains an alternate exon 7, and UFD2a-7/7a, the larger of the 2 isoforms, contains an additional novel exon 7a. Analysis of protein or mRNA expression in mice and zebrafish revealed that a similar pattern of isoform switching occurs during developmental myogenesis of cardiac and skeletal muscle. In vertebrates (humans, rodents, zebrafish), UFD2a-7/7a is expressed only in mature striated muscle. This unique tissue specificity is further validated by the conserved presence of 2 muscle-specific splicing regulatory motifs located in the 3' introns of exons 7 and 7a. UFD2a interacts with VCP/p97, an AAA-type ATPase implicated in processes whose functions appear to be regulated, in part, through their interaction with one or more of 15 previously identified cofactors. UFD2a-7/7a did not interact with VCP/p97 in yeast 2-hybrid experiments, which may allow the ATPase to bind cofactors that facilitate its muscle-specific functions. We conclude that the regulated expression of these UFD2a isoforms most likely imparts divergent functions that are important for myogenisis.

Erythrocyte C4d and complement receptor 1 in systemic lupus erythematosus.

OBJECTIVE: Complement activation and ineffective clearance of complement-bearing immune complexes via erythrocytes contribute to the pathogenesis of systemic lupus erythematosus (SLE). Abnormally high levels of erythrocyte C4d and low levels of complement receptor 1 (CD35) have been reported in SLE and might have diagnostic utility. We investigated whether erythrocyte C4d and complement receptor 1 were specific for SLE and whether there was any association with disease activity. METHODS: Expression of complement receptor 1 (CD35) and complement protein C4d on erythrocytes was measured by indirect immunofluorescence and flow cytometry on the same day as the blood draw, in patients with SLE, patients with rheumatic disease, and in normal controls. RESULTS: Within the SLE population, there was no association with disease activity measured by the physician's global assessment or SELENA SLE Disease Activity Index, nor with past or current lupus nephritis. Assays were not specific for SLE, with higher levels also seen in antiphospholipid syndrome. CONCLUSION: Overlap of erythrocyte C4d and CD35 between SLE and other rheumatic diseases limits their utility as diagnostic tests. However, longitudinal investigation of these assays is warranted, especially given the higher levels in some patients with primary antiphospholipid syndrome.

Asymmetric dimethylarginine is a marker of poor prognosis and coronary calcium in systemic lupus erythematosus.

OBJECTIVE: To determine the association of serum asymmetric dimethylarginine (ADMA) with clinical features, laboratory tests, treatment, cardiovascular risk factors, and subclinical atherosclerosis in patients with systemic lupus erythematosus (SLE). METHODS: Serum ADMA concentrations were determined by ELISA, using purified ADMA as a standard. Coronary calcium was measured by helical computerized tomography. RESULTS: Two hundred patients with SLE participated. Patients had a mean age of 44.3 +/- 11.4 years and were 92% female, 61% Caucasian, 34% African American, 2% Asian, and 2% Hispanic; 18% had elevated ADMA levels. The mean ADMA was 0.31. Significantly higher ADMA levels were found in African Americans (p < 0.001), and were correlated with anti-dsDNA (p < 0.001), anti-Sm (p = 0.005), anti-ribonucleoprotein (p = 0.002), low C4 (p = 0.004), and high erythrocyte sedimentation rate (p < 0.001). ADMA was negatively associated with total cholesterol (p = 0.004). Elevated ADMA was associated with the presence of coronary calcium (p = 0.02). CONCLUSION: Elevated ADMA is strongly associated with African American ethnicity, anti-dsDNA, low complement, and prednisone use, all markers of poor prognosis in SLE. It is negatively associated with hyperlipidemia, but positively associated with coronary calcium. Thus, it identifies a subset of SLE patients with normal lipid levels who are at risk for atherosclerosis.

Soluble CD154 is not associated with atherosclerosis in systemic lupus erythematosus.

OBJECTIVE: Soluble CD154 (sCD154) is involved in the pathogenesis of systemic lupus erythematosus (SLE), as well as in the initiation and progression of atherosclerotic lesions. We determined the association of sCD154 with coronary calcium and carotid plaque at the baseline visit of the Lupus Atherosclerosis Prevention Study. METHODS: Serum samples were assayed for soluble CD154 by ELISA. Coronary calcium was measured by helical computed tomography. Carotid duplex was performed to measure carotid plaque. RESULTS: sCD154 was measured in 183 patients with SLE. Patients had a mean age of 48.8 +/- 10.5 yrs, and 92% were female. Ethnicity included 61% Caucasian, 34% African American, 2% Asian, and 2% Hispanic. sCD154 was not associated with carotid plaque (p = 0.45) nor with coronary calcium (p = 0.43). Indeed, those with carotid plaque had a trend toward lower levels of sCD154 (474 +/- 29.2 vs 526 +/- 5 pg/ml; p = 0.45). CONCLUSION: sCD154 is not associated with subclinical measures of atherosclerosis in SLE, including carotid plaque and coronary calcium.

The MPAC domain is a novel mitotically regulated domain, removed by apoptotic protease cleavage during cell death.

The apoptotic proteases, including caspases and granzyme B, have independent evolutionary origins, yet are both highly specific for cleavage after aspartic acid residues and cleave many of the same substrates at closely spaced sites. In addition, many of these substrates are also reversibly regulated during other processes such as the cell cycle. In these studies, we have identified a novel domain (the MPAC domain: Mitotically Phosphorylated, Apoptotically Cleaved) present at the N-terminus of Ufd2a, which is regulated both by cleavage during cell death, and by phosphorylation during mitosis. We have also identified a corresponding domain, at the C-terminus of polyA polymerase (PAP), which is similarly regulated by phosphorylation during mitosis and is delineated by an apoptotic protease cleavage site. The positioning of the apoptotic cleavage site suggests that it represents a novel connector between the regulatory domain and its functional partner(s), providing insights into the structure and function that guided the evolution of the apoptotic proteases.

A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles.

Carboxypeptidase, vitellogenic-like (CPVL) is a serine carboxypeptidase of unknown function that was first characterized in human macrophages. Initial studies suggested that CPVL is largely restricted to the monocytic lineage, although it may also be expressed by cells outside the immune system. Here, we use a new monoclonal antibody to characterize the properties and localization of CPVL in human macrophages to elucidate a possible function for the protease. CPVL is up-regulated during the maturation of monocytes (MO) to macrophages, although the protein can be seen in both. In primary macrophages, CPVL is glycosylated with high mannose residues and colocalizes with markers for endoplasmic reticulum, while in MO it is more disperse and less clearly associated with endoplasmic reticulum. CPVL is highly expressed in lamellipodia and membrane ruffles, which also concentrate markers of the secretory pathway (MIP-1alpha and tumour necrosis factor-alpha) and major histocompatibility complex (MHC) class I and II molecules. CPVL can be seen on early latex bead and Candida albicans phagosomes, but it is not retained in the maturing phagosome, unlike MHC class I/II. CPVL has a mixed cytosolic and membrane-associated localization but is not detectable on the outer plasma membrane. We propose that CPVL may be involved in antigen processing, the secretory pathway and/or in actin remodelling and lamellipodium formation.

Apoptosis and autoimmunity.

Autoimmune diseases reflect the confluence of genetic, environmental and stochastic events. Recent studies have implicated apoptotic cell death pathways in initiating and propagating autoimmune diseases, as well as in rendering individuals susceptible to such diseases. Similar to autoimmunity, apoptosis is a multistep process, affecting immune and target cells, integrating numerous intrinsic and extrinsic signals, and requiring the actions of multiple gene products. Particularly relevant to the complexity of autoimmunity are the recent observations that apoptotic death might provide a primary source of tolerogen to shape the immune repertoire, or be the target of the immune response in autoimmunity, and that apoptosis is both required for lymphocyte selection and immunoregulation, and is a prominent outcome of immune and inflammatory effector pathways.

Ufd2, a novel autoantigen in scleroderma, regulates sister chromatid separation.

The characterization of molecules recognized by patients with autoimmune diseases has provided significant insights into important biological pathways. In these studies, we define Ufd2 (a novel E4 polyubiquitylating enzyme) as a new autoantigen in scleroderma, and show that it regulates chromosome condensation and separation during mitosis in human cells. Ufd2 is regulated by phosphorylation in mitosis. Inhibition of Ufd2 expression results in mitotic arrest at the metaphase-anaphase transition, where cells manifest abnormal chromosome morphology, missegregated chromosomes, irregular spindles, and premature separation of sister chromatids. This is accompanied by premature separase activation, and accumulation of securin in a novel modified form. We further demonstrate that Ufd2 directly and efficiently ubiquitylates securin in vitro and is required for securin polyubiquitylation in vivo. This is the first description of a physiologic substrate for Ufd2, establishing this E4 enzyme as an important regulator of chromosome condensation and separation during mitosis in human cells. Its targeting in scleroderma, together with many other components of the mitotic machinery, reinforces the concept that mitotic cells may be an important focus of the autoimmune response in this disease.

Immunophenotyping of macrophages in human pulmonary tuberculosis and sarcoidosis.

Classic studies of tuberculosis (TB) revealed morphologic evidence of considerable heterogeneity of macrophages (MØs), but the functional significance of this heterogeneity remains unknown. We have used newly available specific antibodies for selected membrane and secretory molecules to examine the phenotype of MØs in situ in a range of South African patients with TB, compared with sarcoidosis. Patients were human immunodeficiency virus-negative adults and children, and the examined biopsy specimens included lung and lymph nodes. Mature pulmonary MØs (alveolar, interstitial, epithelioid and multinucleated giant cells) selectively expressed scavenger receptor type A and a novel carboxypeptidase-like antigen called carboxypeptidase-related vitellogenin-like MØ molecule (CPVL). CPVL did not display enhanced expression in sarcoidosis, vs. TB patients, as observed with angiotensin-converting enzyme (ACE), a related molecule. Immunocytochemical studies with surfactant proteins (SP)-A and -D showed that type II alveolar cells expressed these collectins, as did MØs, possibly after binding of secreted proteins. Studies with an antibody specific for the C-terminus of fractalkine, a tethered CX3C chemokine, confirmed synthesis of this molecule by bronchiolar epithelial cells and occasional endothelial cells. These studies provide new marker antigens and extend previous studies on MØ differentiation, activation and local interactions in chronic human granulomatous inflammation in the lung.

The human homologue of the yeast polyubiquitination factor Ufd2p is cleaved by caspase 6 and granzyme B during apoptosis.

In the present study, we demonstrate that a human homologue of Ufd2p (a yeast protein that catalyses the formation of long polyubiquitin chains, and is implicated in responses to environmental stress), UFD2 (ubiquitin fusion degradation protein-2), is cleaved during apoptosis induced by multiple stimuli, including UVB irradiation, Fas ligation, staurosporine treatment and cytotoxic lymphocyte granule-induced death. Caspase 6 and granzyme B efficiently cleave UFD2 [k(cat)/K(m)=(4-5) x 10(4) M(-1) x s(-1)] at Asp(123), whereas caspases 3 and 7 cleave UFD2 approx. 10-fold less efficiently immediately upstream at Asp(109). Thus UFD2 is added to the growing list of proteins with closely spaced caspase and granzyme B cleavage sites, suggesting the presence of a previously unrecognized, conserved motif. Both cleavage sites are contained and conserved within a novel 300-amino-acid N-terminal domain present in apparent UFD2 orthologues in mice and zebrafish, but absent in all UFD2 family members in lower eukaryotes. Full-length recombinant UFD2 exhibited ubiquitin-protein ligase ('E3')-like ubiquitination activity in vitro, but this activity was abolished in recombinant UFD2 truncated at the granzyme B/caspase 6 cleavage site. Cleavage of UFD2 by caspases or granzyme B within this putative regulatory N-terminal domain might have important functional consequences within the apoptotic cascade.

Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.