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Mutations of the human valosin-containing protein gene cause autosomal-dominant inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia. We identified strumpellin as a novel valosin-containing protein binding partner. Strumpellin mutations have been shown to cause hereditary spastic paraplegia. We demonstrate that strumpellin is a ubiquitously expressed protein present in cytosolic and endoplasmic reticulum cell fractions. Overexpression or ablation of wild-type strumpellin caused significantly reduced wound closure velocities in wound healing assays, whereas overexpression of the disease-causing strumpellin N471D mutant showed no functional effect. Strumpellin knockdown experiments in human neuroblastoma cells resulted in a dramatic reduction of axonal outgrowth. Knockdown studies in zebrafish revealed severe cardiac contractile dysfunction, tail curvature and impaired motility. The latter phenotype is due to a loss of central and peripheral motoneuron formation. These data imply a strumpellin loss-of-function pathogenesis in hereditary spastic paraplegia. In the human central nervous system strumpellin shows a presynaptic localization. We further identified strumpellin in pathological protein aggregates in inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, various myofibrillar myopathies and in cortical neurons of a Huntington's disease mouse model. Beyond hereditary spastic paraplegia, our findings imply that mutant forms of strumpellin and valosin-containing protein may have a concerted pathogenic role in various protein aggregate diseases.

Conserved ATP-dependent proteases ensure the quality control of mitochondrial proteins and control essential steps in mitochondrial biogenesis. Recent studies demonstrated that non-assembled mitochondrially encoded proteins are degraded to peptides and amino acids that are released from mitochondria. Here, we have characterized peptides extruded from mitochondria by mass spectrometry and identified 270 peptides that are exported in an ATP- and temperature-dependent manner. The peptides originate from 51 mitochondrially and nuclearly encoded proteins localized mainly in the matrix and inner membrane, indicating that peptides generated by the activity of all known mitochondrial ATP-dependent proteases can be released from the organelle. Pulse-labeling experiments in logarithmically growing yeast cells revealed that approximately 6-12% of preexisting and newly imported proteins is degraded and contribute to this peptide pool. Under respiring conditions, we observed an increased proteolysis of newly imported proteins that suggests a higher turnover rate of respiratory chain components and thereby rationalizes the predominant appearance of representatives of this functional class in the detected peptide pool. These results demonstrated a constant efflux of peptides from mitochondria and provided new insight into the stability of the mitochondrial proteome and the efficiency of mitochondrial biogenesis.

The activity of the p53 tumor suppressor protein and the c-Jun protooncogene is regulated by posttranslational modifications, such as phosphorylation or ubiquitination. In addition, covalent attachment of the ubiquitin-like modifier SUMO appears to modulate their transcriptional activity. Sumoylation proceeds via an enzymatic pathway that is mechanistically analogous to ubiquitination, but requires a different E1-activating enzyme and Ubc9, a SUMO-specific E2-conjugating enzyme. Here, we show that two members of the PIAS family, PIAS1 and PIASxbeta, act as specific E3-like ligases that promote sumoylation of p53 and c-Jun in vitro and in vivo. The PIAS proteins physically interact with both p53 and c-Jun. In addition, they bind to Ubc9, suggesting that they recruit the E2 enzyme to their respective substrate. The SUMO ligase activity requires the conserved zinc-finger domain, which is distantly related to the essential RING-finger motif, found in a subset of ubiquitin ligases. Furthermore, similar to RING-type ubiquitin ligases, PIASxbeta can catalyze its own modification. Hence, these data further extend the analogy between the ubiquitin and SUMO pathway. Strikingly, PIAS proteins strongly repress the transcriptional activity of p53, suggesting that the PIAS-SUMO pathway plays a crucial role in the regulation of p53 and presumably other transcription factors.

Autophagy and the ubiquitin proteasome system are the two major processes for the clearance and recycling of proteins and organelles in eukaryotic cells. Evidence is accumulating that there is extensive crosstalk between the two pathways, but the underlying mechanisms are still unclear. We previously found that autophagy 9 (ATG9) and 16 (ATG16) proteins are crucial for full proteasomal activity in the unicellular amoeba Dictyostelium discoideum. In comparison to AX2 wild-type cells, ATG9−and ATG16− cells displayed a 60%, and ATG9−/16− cells a 90%, decrease in proteasomal activity. Mutant cells also showed a significant increase in poly-ubiquitinated proteins and contained large ubiquitin-positive protein aggregates. Here, we focus on possible reasons for these results. Reanalysis of published tandem mass tag-based quantitative proteomic results of AX2, ATG9−, ATG16−, and ATG9−/16− cells revealed no change in the abundance of proteasomal subunits. To identify possible differences in proteasome-associated proteins, we generated AX2 wild-type and ATG16− cells expressing the 20S proteasomal subunit PSMA4 as GFP-tagged fusion protein, and performed co-immunoprecipitation experiments followed by mass spectrometric analysis. The results revealed no significant differences in the abundance of proteasomes between the two strains. However, we found enrichment as well as depletion of proteasomal regulators and differences in the ubiquitination of associated proteins for ATG16−, as compared to AX2 cells. Recently, proteaphagy has been described as a means to replace non-functional proteasomes. We propose that autophagy-deficient D. discoideum mutants suffer from inefficient proteaphagy, which results in the accumulation of modified, less-active, and also of inactive, proteasomes. As a consequence, these cells exhibit a dramatic decrease in proteasomal activity and deranged protein homeostasis. [ABSTRACT FROM AUTHOR]

Background: Before postchemotherapy retroperitoneal lymph node dissection (pcRPLND), in patients with metastasized germ cell tumors (GCTs), those harboring necrosis (NEC) cannot be distinguished from those who have teratoma (TER), resulting in relevant overtreatment, whereas microRNA‐371a‐3p may be predictive for viable GCT. The purpose of this study was to explore messenger RNA (mRNA) and proteins to distinguish TER from NEC in pcRPLND tissue. Methods: The discovery cohort consisted in total of 48 patients, including 16 each with TER, viable GCT, and NEC. Representative areas were microdissected. A NanoString panel and proteomics were used to analyze 770 genes and >5000 proteins. The most significantly and differentially expressed combination of both parameters, mRNA and its associated protein, between TER and NEC was validated using immunohistochemistry (IHC) in an independent validation cohort comprising 66 patients who were not part of the discovery cohort. Results: The authors observed that anterior gradient protein 2 homolog (AGR2) and keratin, type I cytoskeletal 19 (KRT19) were significantly differentially expressed in TER versus NEC in mRNA and protein analyses (proteomics). The technical validation using IHC was successful in the same patients. These proteins were further validated by IHC in the independent patient cohort and exhibited significantly higher levels in TER versus NEC (p <.0001; area under the curve, 1.0; sensitivity and specificity, 100% each). Conclusions: The current study demonstrated that KRT19 and AGR2 mRNA and protein are overexpressed in TER versus NEC in pcRPLND tissue and might serve as a future diagnostic target to detect TER, for instance, by functional imaging, to avoid overtreatment. Plain language summary: The proteins and the corresponding genes called AGR2 and KRT19 can differentiate between teratoma and necrosis in remaining tumor masses after chemotherapy in patients who have metastasized testicular cancer.This may be a way to improve presurgical diagnostics and to reduce the current overtreatment of patients with necrosis only, who could be treated sufficiently by surveillance. Differentially expressed AGR2 and KRT19 can distinguish teratoma from necrosis in postchemotherapy retroperitoneal lymph node dissection tissue on messenger RNA/proteins in patients who have metastasized testicular germ cell tumors. This might be a basis to improve diagnostics and to reduce current overtreatment. [ABSTRACT FROM AUTHOR]

Objective: To analyze the expression of mitochondrial respiratory chain protein subunits in clear cell renal cell carcinoma. Methods: Possible prognostic candidates were determined using The Cancer Genome Atlas database (n = 605). The database provided valid messenger ribonucleic acid expression data for 93 genes encoding for the subunits. Selected subunits were further investigated at the messenger ribonucleic acid and protein level by real‐time polymerase chain reaction, western blot and immunohistochemistry with the cohorts of the University Hospital Bonn. Results: The Cancer Genome Atlas messenger ribonucleic acid expression data indicated univariate and multivariate prognostic impact for seven subunits (NDUFS8, NDUFS7, COX5B, COX6B1, SDHD, COX15 and COX19). Using real‐time polymerase chain reaction, significant downregulation (P < 0.05, n = 74) could be shown for COX5B, COX6B1, NDUFS7 and NDUF8 in clear cell renal cell carcinoma tissue. Survival analysis of polymerase chain reaction data showed a non‐significant relationship (P = 0.067) of high COX5B expression and poor overall survival. Western blot (n = 8) and immunohistochemistry analysis (n = 167) confirmed significant COX5B downregulation on the protein level. Immunohistochemistry analysis identified COX5B as a prognostic marker for overall (P = 0.017) and cancer‐specific survival (P = 0.042). Conclusions: The present study findings suggest downregulation of additional subunits of mitochondrial respiratory chain proteins in clear cell renal cell carcinoma. Remarkably, COX5B, a subunit of the respiratory chain complex IV, can be identified as a novel prognostic marker. [ABSTRACT FROM AUTHOR]

Elementary flux modes (EFMs) emerged as a formal concept to describe metabolic pathways and have become an established tool for constraint-based modeling and metabolic network analysis. EFMs are characteristic (support-minimal) vectors of the flux cone that contains all feasible steady-state flux vectors of a given metabolic network. EFMs account for (homogeneous) linear constraints arising from reaction irreversibilities and the assumption of steady state; however, other (inhomogeneous) linear constraints, such as minimal and maximal reaction rates frequently used by other constraint-based techniques (such as flux balance analysis [FBA]), cannot be directly integrated. These additional constraints further restrict the space of feasible flux vectors and turn the flux cone into a general flux polyhedron in which the concept of EFMs is not directly applicable anymore. For this reason, there has been a conceptual gap between EFM-based (pathway) analysis methods and linear optimization (FBA) techniques, as they operate on different geometric objects. One approach to overcome these limitations was proposed ten years ago and is based on the concept of elementary flux vectors (EFVs). Only recently has the community started to recognize the potential of EFVs for metabolic network analysis. In fact, EFVs exactly represent the conceptual development required to generalize the idea of EFMs from flux cones to flux polyhedra. This work aims to present a concise theoretical and practical introduction to EFVs that is accessible to a broad audience. We highlight the close relationship between EFMs and EFVs and demonstrate that almost all applications of EFMs (in flux cones) are possible for EFVs (in flux polyhedra) as well. In fact, certain properties can only be studied with EFVs. Thus, we conclude that EFVs provide a powerful and unifying framework for constraint-based modeling of metabolic networks. [ABSTRACT FROM AUTHOR]

AMACO (VWA2 protein) is an extracellular matrix protein of unknown function associated with certain basement membranes in skin, lung, and kidney. AMACO is a member of the von Willebrand factor A-like (VWA) domain containing protein superfamily and in addition to three VWA domains it also contains two epidermal growth factor-like domains. One of these contains the rare, overlapping consensus sequences for both O-glucosylation and O-fucosylation. In earlier studies of other proteins the attachment of either core glucose and fucose moieties or of the respective elongated glycans starting with these monosaccharides has been described. By a detailed mass spectrometric analysis we show that both elongated O-glucosylated (Xyl1-3Xyl1 -3Glc) and elongated O-fucosylated glycan chains (NeuAc2-3Gal1-4GlcNAc1-3Fuc) can be attached to AMACO in close proximity on the same epidermal growth factor-like domain. It has been reported that the lack of O-fucosylation can markedly decrease secretion of proteins. However, the secretion of AMACO is not significantly affected when the glycosylation sites are mutated. The number of extracellular matrix proteins carrying the overlapping consensus sequence is very limited and it could be that these modifications have a new, yet unknown function. [ABSTRACT FROM AUTHOR]

OBJECTIVE To immunohistochemically identify the protein composition of adherens junctions, which couple smooth muscle cells mechanically, and to confirm their decrease in different bladder dysfunctions, as studies in geriatric bladder dysfunction show fewer such junctions in patients with detrusor overactivity (DO) and bladder outlet obstruction (BOO). MATERIAL AND METHODS Detrusor biopsies were obtained from video-urodynamically evaluated patients with neurogenic DO (NDO, 31 patients), BOO (six patients) and from six patients with stress urinary incontinence (SUI) with stable, unobstructed detrusors (serving as controls). Specimens were fixed, paraffin-embedded, sectioned, stained with a polyclonal pan-cadherin antibody, monoclonal α-, β- and γ-catenin antibodies, and a monoclonal integrin-β1 antibody. All antibodies were known to react with proteins of adherens junctions. Two examiners unaware of sample origin evaluated the sections qualitatively and using a semiquantitative scale. The results were correlated with the patient groups. RESULTS Specific immunohistochemical staining of pan-cadherin, α-, β- and γ-catenin could not be detected in any detrusor smooth muscle compartment, but was present in the urothelium. There was integrin-β1 reactivity in the basement membranes of bladder smooth muscle cells in 38 of 43 detrusor biopsies. There were no differences among the three groups. CONCLUSION The known proteins of cell-cell adherens junctions are not part of the cell-cell junctions of detrusor smooth muscle. The specific staining of integrin-β1 indicates either the presence of cell-matrix junctions or of cell-cell junctions within the human detrusor. Further studies are needed to identify the complete protein composition of adherens junctions within smooth muscle cells. [ABSTRACT FROM AUTHOR]

The retinoblastoma tumor suppressor protein (pRB) is a major regulator of cell-cycle progression and cellular differentiation. Central to pRB function is the pocket domain, which serves as the main binding region for cellular regulators. In tumors pRB is frequently inactivated by mutations in the pocket domain or by binding of viral oncoproteins to this region. A characteristic feature of these viral oncoproteins and many cellular pRB-binding partners is an LxCxE sequence motif, which interacts with pRB's pocket domain. Here, we show that the ubiquitin-like modifier SUMO is covalently attached to a distinct residue (K720) of pRB within the B-box of the pocket region that binds LxCxE-motif proteins. We provide evidence that SUMO preferentially targets the active, hypophosphorylated form of pRB and show that tumorigenic mutations of pRB in the pocket domain lead to a loss of SUMOylation. Notably, the level of pRB SUMOylation is controlled by the interaction of pRB with viral and cellular LxCxE-motif proteins. Inhibitors of pRB function, including the viral oncoproteins E1A and E7 and the cellular E1A-like inhibitor of differentiation EID-1, completely abolish SUMO modification of pRB. Conversely, pRB mutants deficient in binding of LxCxE-motif proteins exhibit a drastically enhanced modification by SUMO. Finally, we provide evidence that SUMOylation can influence pRB function, as the SUMO-deficient pRBK720R mutant exerts a slightly higher repressive potential on an E2F-responsive reporter gene than wild-type pRB. Taken together, these data identify SUMO modification as a novel post-translational modification of pRB that may control pRB activity by modulating LxCxE-pocket interactions.Oncogene (2005) 24, 3810-3818. doi:10.1038/sj.onc.1208539 Published online 4 April 2005 [ABSTRACT FROM AUTHOR]

Post-translational modification with the ubiquitin-like SUMO protein is involved in the regulation of many cellular key processes. The SUMO system modulates signal transduction pathways, including cytokine, Wnt, growth factor and steroid hormone signalling. SUMO frequently restrains the activity of downstream transcription factors in these pathways presumably by facilitating the recruitment of corepressors or mediating the assembly of repressor complexes. Additionally, evidence is accumulating that SUMO controls pathways important for the surveillance of genome integrity. SUMO regulates the PML/p53 tumour suppressor network, a key determinant in the cellular response to DNA damage. Moreover, proteins that maintain genomic stability by functioning at the interface between DNA replication, recombination and repair processes undergo SUMOylation. We will discuss some key findings that exemplify the role of SUMO in transcriptional regulation and genome surveillance.Oncogene (2004) 23, 1998-2008. doi:10.1038/sj.onc.1207415 [ABSTRACT FROM AUTHOR]

Examines the integration of G[sub 12] and G[sub 13] heterotrimeric G-proteins signaling pathways. Role of G[sub 13] on blood vessel development; Mediation of cell migratory response to lysophosphatidic acid in embryonic fibroblast cells; Use of immunohistochemistry analysis.

Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO regulates various cellular processes, such as nuclear transport, signal transduction, stress response and cell-cycle progression. But, in contrast to ubiquitylation, sumoylation does not tag proteins for degradation, but seems to enhance their stability or modulate their subcellular compartmentalization. [ABSTRACT FROM AUTHOR]

The PML protein, identified first as part of the oncogenic PML­RARα chimera in acute promyelocytic leukemia (APL), concentrates within discrete subnuclear structures, corresponding to some types of nuclear bodies. These structures are disrupted in APL cells, and retinoic acid (RA) can trigger their reorganization, correlating with its therapeutic effect in this type of leukemia. Recently, arsenic trioxide (As2O3) was identified as a potent antileukemic agent which, similarly to RA, induces complete remissions in APL patients. Here we show that, in APL cells, As2O3 triggers rapid degradation of PML—RARα and provokes the restoration of intact nuclear bodies. In non-APL cells, the ubiquitin-like protein SUMO-1 is covalently attached to a subset of wild-type PML in a reversible and phosphorylation-dependent manner. The unmodified form of PML is found in the soluble nucleoplasmic fraction, whereas the SUMO-1-polymodified forms of PML are compartmentalized exclusively in the PML nuclear bodies. As2O3 administration strikingly increases the pool of SUMO-1—PML conjugates that, subsequently, accumulate in enlarged nuclear bodies. In contrast to PML—RARα, the overall amount of PML seems to remain unaltered up to 36 h following As2O3 treatment. These findings indicate that the conjugation of PML with SUMO-1 modulates its intracellular localization and suggest that post-translational modification by SUMO-1 may be more generally involved than previously suspected in the targeting of proteins to distinct subcellular structures. They provide additional evidence that the role of ‘ubiquitin-like’ post-translational modification is not limited to a degradation signal. [ABSTRACT FROM AUTHOR]

In this issue of Genes & Development, Yang and colleagues (pp. 1847-1858) identify new components of a small ubiquitin-like modifier (SUMO)-like interaction network that orchestrates and fine-tunes the Fanconi anemia (FA) pathway and replication-coupled repair. This new pathway emphasizes the intricate interplay of ubiquitin (Ub) and SUMO networks in the DNA damage response. [ABSTRACT FROM AUTHOR]

Recently, an alternative route to the proteasomal protein-degradation pathway was discovered that specifically targets transmembrane proteins marked with a single ubiquitin to the endosomal multivesicular body (MVB) and, subsequently, to the vacuole (yeast) or lysosome (animals), where they are degraded by proteases. Vps23p/TSG101 is a key component of the ESCRT I-III machinery in yeast and animals that recognizes mono-ubiquitylated proteins and sorts them into the MVB. Here, we report that the Arabidopsis ELCH (ELC) gene encodes a Vps23p/TSG101 homolog, and that homologs of all known ESCRT I-III components are present in the Arabidopsis genome. As with its animal and yeast counterparts, ELC binds ubiquitin and localizes to endosomes. Gel-filtration experiments indicate that ELC is a component of a high-molecular-weight complex. Yeast two-hybrid and immunoprecipitation assays showed that ELC interacts with Arabidopsis homologs of the ESCRT I complex. The elc mutant shows multiple nuclei in various cell types, indicating a role in cytokinesis. Double-mutant analysis with kaktus shows that increased ploidy levels do not influence the cytokinesis effect of elc mutants, suggesting that ELC is only important during the first endoreduplication cycle. Double mutants with tubulin folding cofactor a mutants show a synergistic phenotype, suggesting that ELC regulates cytokinesis through the microtubule cytoskeleton. [ABSTRACT FROM AUTHOR]

Recombinant forms of the glycoprotein TSC-36/Flik were expressed in human cells and used to compare their structural and functional properties with those described for other members of the BM-40/SPARC/osteonectin protein family. TSC-36 was found to occur in two charge isoforms that differ in the extent of sialylation of otherwise identical N-linked, complex type oligosaccharides. Conformational analysis with both circular dichroism and intrinsic fluorescence spectroscopy showed a lack of significant structural changes upon calcium addition or depletion. This finding is in contrast to results obtained for several other BM-40 family members and indicates that the extracellular calcium-binding domain in TSC-36 is non-functional. The lack of conservation of important functional features common to several other members of the BM-40 family indicates that TSC-36, despite its sequence homology to BM-40, has evolved clearly distinct properties. [ABSTRACT FROM AUTHOR]

Epigenetic memory mediated by Polycomb group (PcG) proteins must be maintained during cell division, but must also be flexible to allow cell fate transitions. Here we quantify dynamic chromatin-binding properties of PH::GFP and PC::GFP in living Drosophila in two cell types that undergo defined differentiation and mitosis events. Quantitative fluorescence recovery after photobleaching (FRAP) analysis demonstrates that PcG binding has a higher plasticity in stem cells than in more determined cells and identifies a fraction of PcG proteins that binds mitotic chromatin with up to 300-fold longer residence times than in interphase. Mathematical modeling examines which parameters best distinguish stem cells from differentiated cells. We identify phosphorylation of histone H3 at Ser 28 as a potential mechanism governing the extent and rate of mitotic PC dissociation in different lineages. We propose that regulation of the kinetic properties of PcG-chromatin binding is an essential factor in the choice between stability and flexibility in the establishment of cell identities. [ABSTRACT FROM AUTHOR]

Abstract: Background: We investigated the role of the programmed cell death 4 (PDCD4) tumor suppressor gene in specimens of transitional cell carcinoma and of healthy individuals. Methods: PDCD4 immunohistochemical expression was investigated in 294 cases in histologically proven transitional cell carcinoma in different tumorous stages (28 controls, 122 non-muscle invasive urothelial carcinoma, stages Tis-T1, 119 invasive transitional cell carcinoma stages T2–T4 and 25 metastases). MiR-21 expression, an important PDCD4 regulator, was assessed with real-time PCR analysis and showed inverse correlation to tissue PDCD4 expression. Results: Nuclear and cytoplasmatic PDCD4 immunostaining decreased significantly with histopathological progression of the tumor (p <0001). Controls showed strong nuclear and cytoplasmatic immunohistochemical staining. MiR-21 up regulation in tissue corresponded to PDCD4 suppression. Conclusions: These data support a decisive role for PDCD4 down regulation in transitional cell carcinoma and confirm miR-21 as a negative regulator for PDCD4. Additionally, PDCD4 immunohistochemical staining turns out to be a possible diagnostic marker for transitional cell carcinoma. [Copyright &y& Elsevier]

Mitochondria harbor a conserved proteolytic system that mediates the complete degradation of organellar proteins. ATP-dependent proteases, like a Lon protease in the matrix space and m- and i-AAA proteases in the inner membrane, degrade malfolded proteins within mitochondria and thereby protect the cell against mitochondrial damage. Proteolytic breakdown products include peptides and free amino acids, which are constantly released from mitochondria. It remained unclear, however, whether the turnover of malfolded proteins involves only ATP-dependent proteases or also oligopeptidases within mitochondria. Here we describe the identification of Mop112, a novel metallopeptidase of the pitrilysin family M16 localized in the intermembrane space of yeast mitochondria. This peptidase exerts important functions for the maintenance of the respiratory competence of the cells that overlap with the i-AAA protease. Deletion of MOP112 did not affect the stability of misfolded proteins in mitochondria, but resulted in an increased release from the organelle of peptides, generated upon proteolysis of mitochondrial proteins. We find that the previously described metallopeptidase saccharolysin (or Prd1) exerts a similar function in the intermembrane space. The identification of peptides released from peptidase-deficient mitochondria by mass spectrometry indicates a dual function of Mop112 and saccharolysin: they degrade peptides generated upon proteolysis of proteins both in the intermembrane and matrix space and presequence peptides cleaved off by specific processing peptidases in both compartments. These results suggest that the turnover of mitochondrial proteins is mediated by the sequential action of ATP-dependent proteases and oligopeptidases, some of them localized in the intermembrane space. [ABSTRACT FROM AUTHOR]

The SP-RING or Miz zinc finger domain that is related to the classical RING-finger motif, defines a class of proteins that can act as E3-like factors in the pathway of small ubiquitin-related modifier (SUMO) conjugation. This family includes the mammalian protein inhibitor of activated STAT (PIAS) proteins and related proteins from lower eukaryotes. Here we report the existence of a gene in Leishmania infantum, present as two identical copies placed upstream of each MAT2 gene copy, and transcribed as a single ∼2.2 kb mRNA both in the logarithmic and stationary phases of the promastigote stage. This gene encodes a 47 kDa protein that has been named LORIEN. LORIEN is circumscribed to the cell periphery and it is antigenic during L. infantum infection of dogs and hamsters. Strikingly, this novel protein contains a highly conserved SP-RING/Miz zinc finger domain, raising the possibility that a SUMO or ubiquitin-like system may exist in this microorganism. [Copyright &y& Elsevier]

Mutations in the SALL1 gene on chromosome 16q12.1 cause Townes–Brocks syndrome (TBS). This autosomal dominantly inherited disorder is characterized by typical malformations of the thumbs, the ears, and the anus, and also commonly affects the kidneys and other organ systems. SALL1 has recently been shown to localize to chromocenters and other heterochromatin foci in murine fibroblasts and to interact with the telomere-repeat-binding factor TRF1/PIN2. Here, we show that the ubiquitin-conjugating enzyme 2I (UBE2I), the human homolog of S. cerevisiae UBC9, and the small ubiquitin-like modifier-1 (SUMO-1) interact with SALL1 in the yeast two-hybrid system. The interaction of SALL1 and UBE2I was confirmed in a glutathione S-transferase (GST) pull-down experiment. In an in vitro assay, it could be demonstrated that SALL1 is covalently modified by at least two SUMO-1 molecules in the presence of UBA2/AOS1 and UBE2I. Mutation of lysine 1086 of SALL1 to arginine abrogates SALL1 sumoylation, suggesting the presence of a polymeric SUMO-1 chain in the wild type state. [Copyright &y& Elsevier]