Philip A. Robinson

E3 ubiquitin ligases

The selectivity of the ubiquitin-26 S proteasome system (UPS) for a particular substrate protein relies on the interaction between a ubiquitin-conjugating enzyme (E2, of which a cell contains relatively few) and a ubiquitin-protein ligase (E3, of which there are possibly hundreds). Post-translational modifications of the protein substrate, such as phosphorylation or hydroxylation, are often required prior to its selection. In this way, the precise spatio-temporal targeting and degradation of a given substrate can be achieved. The E3s are a large, diverse group of proteins, characterized by one of several defining motifs. These include a HECT (homologous to E6-associated protein C-terminus), RING (really interesting new gene) or U-box (a modified RING motif without the full complement of Zn2+-binding ligands) domain. Whereas HECT E3s have a direct role in catalysis during ubiquitination, RING and U-box E3s facilitate protein ubiquitination. These latter two E3 types act as adaptor-like molecules. They bring an E2 and a substrate into sufficiently close proximity to promote the substrates ubiquitination. Although many RING-type E3s, such as MDM2 (murine double minute clone 2 oncoprotein) and c-Cbl, can apparently act alone, others are found as components of much larger multi-protein complexes, such as the anaphase-promoting complex. Taken together, these multifaceted properties and interactions enable E3s to provide a powerful, and specific, mechanism for protein clearance within all cells of eukaryotic organisms. The importance of E3s is highlighted by the number of normal cellular processes they regulate, and the number of diseases associated with their loss of function or inappropriate targeting.

Doha agreement meeting on terminology and definitions in groin pain in athletes

Background Heterogeneous taxonomy of groin injuries in athletes adds confusion to this complicated area. Aim The ‘Doha agreement meeting on terminology and definitions in groin pain in athletes’ was convened to attempt to resolve this problem. Our aim was to agree on a standard terminology, along with accompanying definitions. Methods A one-day agreement meeting was held on 4 November 2014. Twenty-four international experts from 14 different countries participated. Systematic reviews were performed to give an up-to-date synthesis of the current evidence on major topics concerning groin pain in athletes. All members participated in a Delphi questionnaire prior to the meeting. Results Unanimous agreement was reached on the following terminology. The classification system has three major subheadings of groin pain in athletes: 1. Defined clinical entities for groin pain: Adductor-related, iliopsoas-related, inguinal-related and pubic-related groin pain. 2. Hip-related groin pain. 3. Other causes of groin pain in athletes. The definitions are included in this paper. Conclusions The Doha agreement meeting on terminology and definitions in groin pain in athletes reached a consensus on a clinically based taxonomy using three major categories. These definitions and terminology are based on history and physical examination to categorise athletes, making it simple and suitable for both clinical practice and research.

Adductor-related groin pain in competitive athletes. Role of adductor enthesis, magnetic resonance imaging, and entheseal pubic cleft injections.

BACKGROUND Adductor dysfunction is a condition that can cause groin pain in competitive athletes, but the source of the pain has not been established and no specific interventions have been evaluated. We previously defined a magnetic resonance imaging protocol to visualize adductor enthesopathy. The aim of this study was to elucidate, in the context of adductor-related groin pain in the competitive athlete, the role of the adductor enthesis (origin), the relevance of adductor enthesopathy diagnosed with magnetic resonance imaging, and the efficacy of entheseal pubic cleft injections of local anesthetic and steroids. METHODS We reviewed the findings in a consecutive series of twenty-four competitive athletes who had presented to our sports medicine clinic with groin pain secondary to adductor longus dysfunction. Magnetic resonance imaging was performed to assess the adductor longus origin for the presence or absence of enthesopathy. Seven patients (Group 1) had no evidence of enthesopathy on magnetic resonance imaging, and seventeen patients (Group 2) had enthesopathy confirmed on magnetic resonance imaging. All patients were treated with a single pubic cleft injection of local anesthetic and steroid into the adductor enthesis. At one year after this treatment, the patients were assessed for recurrence of symptoms. RESULTS On clinical reassessment five minutes after the injection, all twenty-four athletes reported resolution of the groin pain. At one year, none of the seven patients in Group 1 had experienced a recurrence. Sixteen of the seventeen patients in Group 2 had a recurrence of the symptoms (p < 0.001) at a mean of five weeks (range, one to sixteen weeks) after the injection. CONCLUSIONS A single entheseal pubic cleft injection can be expected to afford at least one year of relief of adductor-related groin pain in a competitive athlete with normal findings on a magnetic resonance imaging scan; however, it should be employed only as a diagnostic test or short-term treatment for a competitive athlete with evidence of enthesopathy on magnetic resonance imaging.

The Ubiquitin-conjugating Enzymes UbcH7 and UbcH8 Interact with RING Finger/IBR Motif-containing Domains of HHARI and H7-AP1

Ubiquitinylation of proteins appears to be mediated by the specific interplay between ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s). However, cognate E3s and/or substrate proteins have been identified for only a few E2s. To identify proteins that can interact with the human E2 UbcH7, a yeast two-hybrid screen was performed. Two proteins were identified and termed human homologue of Drosophila ariadne (HHARI) and UbcH7-associated protein (H7-AP1). Both proteins, which are widely expressed, are characterized by the presence of RING finger and in between RING fingers (IBR) domains. No other overt structural similarity was observed between the two proteins. In vitrobinding studies revealed that an N-terminal RING finger motif (HHARI) and the IBR domain (HHARI and H7-AP1) are involved in the interaction of these proteins with UbcH7. Furthermore, binding of these two proteins to UbcH7 is specific insofar that both HHARI and H7-AP1 can bind to the closely related E2, UbcH8, but not to the unrelated E2s UbcH5 and UbcH1. Although it is not clear at present whether HHARI and H7-AP1 serve, for instance, as substrates for UbcH7 or represent proteins with E3 activity, our data suggests that a subset of RING finger/IBR proteins are functionally linked to the ubiquitin/proteasome pathway.

UCH‐L1 aggresome formation in response to proteasome impairment indicates a role in inclusion formation in Parkinson's disease

Aggresomes are associated with many neurodegenerative disorders, including Parkinsons disease, and polyglutamine disorders such as Huntingtons disease. These inclusions commonly contain ubiquitylated proteins. The stage at which these proteins are ubiquitylated remains unclear. A malfunction of the ubiquitin/proteasome system (UPS) may be associated with their formation. Conversely, it may reflect an unsuccessful attempt by the cell to remove them. Previously, we demonstrated that overexpression of Parkin, a ubiquitin‐protein ligase associated with autosomal recessive juvenile Parkinsonism, generates aggresome‐like inclusions in UPS compromised cells. Mutations in the de‐ubiquitylating enzyme, UCH‐L1, cause a rare form of Parkinsonism. We now demonstrate that overexpression of UCH‐L1 also forms ribbon‐like aggresomes in response to proteasomal inhibition. Disease‐associated mutations, which affect enzymatic activities, significantly increased the number of inclusions. UCH‐L1 aggresomes co‐localized with ubiquitylated proteins, HSP70, γ‐tubulin and, to a lesser extent, the 20S proteasome and the chaperone BiP. Similar to Parkin inclusions, we found UCH‐L1 aggresomes to be surrounded by a tubulin rather than a vimentin cage‐like structure. Furthermore, UCH‐L1 aggregates with Parkin and α‐synuclein in some, but not all inclusions, suggesting the heterogeneous nature of these inclusion bodies. This study provides additional evidence that aggregation‐prone proteins are likely to recruit UPS components in an attempt to clear proteins from failing proteasomes. Furthermore, UCH‐L1 accumulation is likely to play a pathological role in inclusion formation in Parkinsons disease.

cDNA Array Analysis of cag Pathogenicity Island-Associated Helicobacter pylori Epithelial Cell Response Genes

ABSTRACT Helicobacter pylori strains containing thecag pathogenicity island (PAI) induce NF-κB activation and interleukin-8 secretion in gastric epithelial cells. The aim of this study was to investigate changes in epithelial gene expression induced by cag PAI-positive and -negative strains ofH. pylori using high-density cDNA array hybridization technology. Radio-labeled cDNA prepared from H. pylori-infected Kato 3 gastric epithelial cells was hybridized to high-density cDNA arrays to identify changes in epithelial gene expression compared to noninfected controls. In vivo expression of selected, differentially expressed genes was examined by reverse transcription-PCR analysis of H. pylori-positive and -negative gastric mucosa. Screening of ca. 57,800 cDNAs identified 208 known genes and 48 novel genes and/or expressed sequence tags of unknown function to be differentially expressed in Kato 3 cells following H. pylori infection. Marked differences in gene expression profiles were observed following cagPAI-positive and cag PAI-negative infection with 15 novel cDNAs and 92 known genes being differentially expressed. H. pylori was found to change the expression of genes encoding growth factors and cytokine/chemokines and their receptors, apoptosis proteins, transcription factors and metalloprotease-disintegrin proteins (ADAMs), and tissue inhibitors of metalloproteinases. Gastric differential expression of selected known genes (amphiregulin and ADAM 10) and a novel gene (HPYR1) was confirmed in vivo in patients with H. pylori infection. Confirmation of the in vivo expression of selected genes demonstrates the usefulness of this approach for investigating pathogen-induced changes in host gene expression.

A novel presenilin-2 splice variant in human Alzheimer's disease brain tissue.

Abstract: Mutations in the presenilin‐1 (PS‐1) and presenilin‐2 (PS‐2) genes account for the majority of cases of early‐onset familial Alzheimers disease (AD). Alternative splicing forms of the PS‐1 and PS‐2 gene products have previously been reported in fibroblast and brain tissue from both familial and sporadic AD patients, as well as from normal tissues and cell lines. We demonstrate here unusual alternative splicing of the PS‐2 gene that leads to the generation of mRNA lacking exon 5 in human brain tissue. This product was more frequently detected in brain tissue from sporadic AD patients (70.0%; 21 of 30) than from normal age‐matched controls (17.6%; three of 17). In cultured neuroblastoma cells, this splice variant was generated in hypoxia but not under other forms of cellular stress. Hypoxia‐mediated induction of this splice variant was blocked by pretreatment of neuroblastoma cells with the protein synthesis inhibitor cycloheximide or antioxidants such as N‐acetylcysteine and diphenyl iodonium, suggesting that hypoxia‐mediated oxidant stress might, at least in part, underlie the alternative splicing of PS‐2 mRNA through de novo protein synthesis. Furthermore, the stable transfectants of this splice variant produced the N‐terminal part of PS‐2 protein (15 kDa) and were more susceptible to cellular stresses than control transfectants. These results suggest the possibility that altered presenilin gene products in stress conditions may also participate in the pathogenesis of AD.

Expression of Interleukin-18, a Th1 Cytokine, in Human Gastric Mucosa Is Increased in Helicobacter pylori Infection

Interleukin-18 (IL-18), a cytokine that promotes Th1 responses, is processed to the active mature protein by caspase-1. The effects of Helicobacter pylori infection on gastric IL-18 and caspase-1 were examined. In antral mucosa, IL-18 mRNA expression was greater (P<.01) in H. pylori-positive (n=40) than in H. pylori-negative patients (n=29) with normal mucosa. Inactive precursor (24 kDa) and mature (18 kDa) IL-18 were present in antral biopsy specimens from uninfected and infected subjects. In corpus mucosa, mature IL-18 and a 16-kDa protein, corresponding to inactive IL-18, were present. Active caspase-1 p20 subunit was detected in antral and corpus mucosa of infected and uninfected subjects. These data show that, although H. pylori infection is associated with increased antral IL-18 mRNA expression, mature IL-18 protein and active caspase-1 p20 are present in mucosa of both H. pylori-infected and -uninfected subjects. IL-18 may have an important role in promoting gastric Th1 responses in H. pylori infection.

Gastric mucosal cytokine and epithelial cell responses to Helicobacter pylori infection in Mongolian gerbils

Experimental infection with Helicobacter pylori in Mongolian gerbils results in chronic gastritis and gastric cancer. To investigate epithelial cell proliferation, apoptosis, and mucosal cytokine responses in gastritis, Mongolian gerbils were infected with the H pylori SS1 strain. At 4 weeks post‐infection, gastritis was predominantly within the antrum, but extended to the corpus in approximately 50% of gerbils by 36 weeks. Epithelial cell proliferation and apoptosis in glandular epithelial cells were increased with infection. Antral cell proliferation, but not apoptosis, correlated significantly with gastric inflammation. In female gerbils, H pylori significantly increased expression of transcripts for IFN‐γ and IL‐12p40, but not TGF‐β or IL‐10, in the gastric mucosa. Significantly reduced IFN‐γ and IL‐12p40 responses were observed in male gerbils infected with H pylori, but epithelial proliferative and apoptotic responses were comparable to those of females. These studies demonstrate that the female gerbil cytokine response to H pylori has a Th1 profile and that there are gender differences in the magnitude of the gastric cytokine responses to H pylori. The absence of a down‐regulatory cytokine response may account for the more severe gastritis observed with H pylori infection in gerbils than in mice. Copyright

Cadaveric and MRI Study of the Musculotendinous Contributions to the Capsule of the Symphysis Pubis

OBJECTIVE The purpose of this article is to define the relations of the symphysis pubis and capsular tissues to the adductor and rectus abdominis soft-tissue attachments on cadaver dissection and correlate with MRI of the anterior pelvis. SUBJECTS AND METHODS Seventeen cadavers (8 males and 9 females; mean age, 80 years) were dissected bilaterally. Rectus abdominis and adductor muscles were traced to the pubis and further attachments to the pubic symphysis were defined. Ten asymptomatic (mean age, 17; age range, 16.5-29 years) male athletes underwent 1.5-T MRI of the anterior pelvis with two surface microcoils (each 42 mm in diameter). An axial T2-weighted turbo spin-echo (TSE) sequence (TR/TE, 2,609/106; voxel size, 0.4 mm) was obtained. Axial and sagittal 3D T1-weighted fast-field echo (FFE) sequences (25/4.9; voxel size, 0.3 mm) were obtained. Sequences were repeated incorporating fat suppression and i.v. gadolinium. The relation of the symphysis pubis, disk, and capsular tissues to the insertions of the rectus abdominis, adductor muscles, and gracilis were independently evaluated by two experienced radiologists blinded to all clinical details. RESULTS In all 17 cadaver specimens, the adductor longus and rectus abdominis attached to the capsule and disk of the symphysis pubis, whereas the adductor brevis had an attachment to the capsule in seven specimens and the gracilis in one. All adductor tendons attached to the pubis. In all 10 athletes, the adductor longus and rectus abdominis bilaterally contributed to the capsular tissues and disk. This was only the case for the adductor brevis in four athletes. No other tendons involved the capsular tissues. CONCLUSION Cadaver and MRI findings show an intimate relationship between the adductor longus; rectus abdominis; and symphyseal cartilage, disk, and capsular tissues.