Post-translational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr90 mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2T90K modifed proteins from human cell lysates produced a diGly remnant on SUMO2T90K conjugated lysines enabling immunoprecipitation of SUMO2T90K modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO enriched peptides revealed over 1,000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and potential other similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.
The genotoxin cisplatin is commonly used in chemotherapy to treat solid tumors, yet our understanding of the mechanism underlying the drug response is limited. In a focused siRNA screen, using an siRNA library targeting genes involved in ubiquitin and ubiquitin-like signaling, we identified the E3 ubiquitin ligase HOIP as a key regulator of cisplatin-induced genotoxicity. HOIP forms, with SHARPIN and HOIL-1L, the linear ubiquitin assembly complex (LUBAC). We show that cells deficient in the HOIP ligase complex exhibit hypersensitivity to cisplatin. This is due to a dramatic increase in caspase 8/caspase 3 mediated apoptosis that is strictly dependent on ATM-, but not ATR-mediated DNA damage checkpoint activation. Moreover, basal and cisplatin-induced activity of the stress response kinase JNK is enhanced in HOIP-depleted cells and, conversely, JNK inhibition can increase cellular resistance to cisplatin and reverse the apoptotic hyper-activation in HOIP-depleted cells. Furthermore, we show that HOIP depletion sensitizes cancer cells, derived from carcinomas of various origins, through an enhanced apoptotic cell death response. We also provide evidence that ovarian cancer cells classified as cisplatin-resistant can regain sensitivity following HOIP down-regulation. Cumulatively, our study identifies a HOIP-regulated anti-apoptotic signaling pathway, and we envisage HOIP as a potential target for the development of combinatorial chemotherapies to potentiate the efficacy of platinum-based anti-cancer drugs.
siRNA screen; cisplatin; HOIP; apoptosis; ubiquitin-signaling
This research was done to assess levels of psychosocial stress and related hazards [(burnout, depression, and posttraumatic stress disorder (PTSD)] among emergency medical responders (EMRs).
Materials and Methods:
A comparative cross-sectional study was conducted upon (140) EMRs and a comparative group composed of (140) nonemergency workers. The groups studied were subjected to semistructured questionnaire including demographic data, survey for job stressors, Maslach burn out inventory (MBI), Beck depression inventory (BDI), and Davidson Trauma scale for PTSD.
The most severe acute stressors among EMRs were dealing with traumatic events (88.57%), followed by dealing with serious accidents (87.8%) and young victims (87.14%). Chronic stressors were more commonly reported among EMRs with statistically significant differences (P < 0.05) except for social support with colleagues and supervisors. EMRs had statistically significant higher levels of emotional exhaustion (EE) (20%) and depersonalization (DP) (9.3%) compared with comparative group (4.3%, 1.4% respectively). Also, there was no statistically significant difference between two groups as regards lower personal achievement or depression symptoms (P > 0.05). There was increased risk of PTSD for those who had higher stress levels from death of colleagues [odds ratio (OR) [95% confidence interval (CI)] = 2.2 (0.7-7.6), exposure to verbal or physical assault OR (95% CI) = 1.6 (0.5-4.4) and dealing with psychiatric OR (95% CI) 1.4 (0.53.7) (P > 0.05)
EMRs group had more frequent exposure to both acute and chronic work-related stressors than comparative group. Also, EMRs had higher levels of EE, DP, and PTSD compared with comparative group. EMRs are in need for stress management program for prevention these of stress related hazards on health and work performance.
Burnout; depression; emergency medical responders; post-traumatic stress disorders; psychosocial stress among emergency medical responders; work-related stress among ambulance workers
The System L1-type amino acid transporter mediates transport of large neutral amino acids (LNAA) in many mammalian cell-types. LNAA such as leucine are required for full activation of the mTOR-S6K signalling pathway promoting protein synthesis and cell growth. The SLC7A5 (LAT1) catalytic subunit of high-affinity System L1 functions as a glycoprotein-associated heterodimer with the multifunctional protein SLC3A2 (CD98). We generated a floxed Slc7a5 mouse strain which, when crossed with mice expressing Cre driven by a global promoter, produced Slc7a5 heterozygous knockout (Slc7a5+/−) animals with no overt phenotype, although homozygous global knockout of Slc7a5 was embryonically lethal. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout (MS-Slc7a5-KO) mice were used to study the role of intracellular LNAA delivery by the SLC7A5 transporter for mTOR-S6K pathway activation in skeletal muscle. Activation of muscle mTOR-S6K (Thr389 phosphorylation) in vivo by intraperitoneal leucine injection was blunted in homozygous MS-Slc7a5-KO mice relative to wild-type animals. Dietary intake and growth rate were similar for MS-Slc7a5-KO mice and wild-type littermates fed for 10 weeks (to age 120 days) with diets containing 10%, 20% or 30% of protein. In MS-Slc7a5-KO mice, Leu and Ile concentrations in gastrocnemius muscle were reduced by ∼40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in muscles from MS-Slc7a5-KO mice, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MS-Slc7a5-KO mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signalling in mice, although it appears non-essential (or is sufficiently compensated by e.g. SLC7A8 (LAT2)) for maintenance of normal muscle mass.
Aspergillus fumigatus is the causative agent of IA (invasive aspergillosis) in immunocompromised patients. It possesses a cell wall composed of chitin, glucan and galactomannan, polymeric carbohydrates synthesized by processive glycosyltransferases from intracellular sugar nucleotide donors. Here we demonstrate that A. fumigatus possesses an active AfAGM1 (A. fumigatus N-acetylphosphoglucosamine mutase), a key enzyme in the biosynthesis of UDP (uridine diphosphate)–GlcNAc (N-acetylglucosamine), the nucleotide sugar donor for chitin synthesis. A conditional agm1 mutant revealed the gene to be essential. Reduced expression of agm1 resulted in retarded cell growth and altered cell wall ultrastructure and composition. The crystal structure of AfAGM1 revealed an amino acid change in the active site compared with the human enzyme, which could be exploitable in the design of selective inhibitors. AfAGM1 inhibitors were discovered by high-throughput screening, inhibiting the enzyme with IC50s in the low μM range. Together, these data provide a platform for the future development of AfAGM1 inhibitors with antifungal activity.
cell wall; drug target; enzyme; inhibitor; nucleotide sugar; protein structure; AfAGM1, A. fumigatus N-acetylphosphoglucosamine mutase; AGM1, N-acetylphosphoglucosamine mutase; CaAGM1, Candida albicans AGM1; Fru-6P, fructose 6-phosphate; G6PDH, glucose-6-phosphate dehydrogenase; GlcNAc, N-acetylglucosamine; GlcNAc-1P, N-acetylglucosamine-1-phosphate; GlcN-6P, glucosamine 6-phosphate; GFA1, glutamine: Fru-6P amidotransferase; GNA1, GlcN-6P acetyltransferase; IA, invasive aspergillosis; MIC, minimum inhibitory concentration; MM, minimal medium; RMSD, root mean square deviation; UAP1, UDP–GlcNAc pyrophosphorylase; UDP, uridine diphosphate
HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to
hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response
to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the
present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52
(ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the
HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and
resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR
(untranslated region)-dependent poly(A)-tail-length-independent destabilization in
HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body
(processing body) components and we confirmed further that USP52 protein and HIF1A
mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of
GW182 or LSM1 resulted in a reduction of HIF1A
mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA
stability, and demonstrate that USP52 is a key component of P-bodies required to prevent
HIF1A mRNA degradation.
AU-rich element (ARE)-mediated degradation (AMD); hypoxia-inducible factor 1α (HIF1A); poly(A) nuclease 2 (PAN2); processing body (P-body); pseudo-deubiquitinating enzyme (pseudo-DUB); ubiquitin-specific protease 52 (USP52); aHIF, antisense hypoxia-inducible factor; ARE, AU-rich element; AMD, ARE-mediated degradation; CA9, carbonic anhydrase IX; CHX, cycloheximide; CTNNB1, β-catenin; CUL2, cullin 2; DCP1A, decapping enzyme 1A; DUB, deubiquitinating enzyme; ERG, Ets-related gene; FBS, fetal bovine serum; FISH, fluorescent in situ hybridization; GFP, green fluorescent protein; GLUT1, glucose transporter 1; HEK, human embryonic kidney; HIF1A, hypoxia-inducible factor 1α; HIF1B, hypoxia-inducible factor 1β; HRE, hypoxia-response element; LC, liquid chromatography; LDHA, lactate dehydrogenase A; miRNA, microRNA; MS/MS, tandem MS; NEDD8, neural-precursor-cell-expressed developmentally down-regulated 8; NP-40, Nonidet P40; NT, Non-Targeting; PABPC1, poly(A)-binding protein C1; PAN2, poly(A) nuclease 2; P-body, processing body; PHD, prolyl hydroxylase; RT, reverse transcription; siRNA, short interfering RNA; TCE, transcription elongation factor; TRIM21, tripartite motif-containing 21; TTP, tristetrapolin; USP52, ubiquitin-specific protease 52; UTR, untranslated region; VEGF, vascular endothelial growth factor; VHL, von Hippel–Lindau; YFP, yellow fluorescent protein
Natural products are often large, synthetically intractable molecules, yet frequently offer surprising inroads into previously unexplored chemical space for enzyme inhibitors. Argifin is a cyclic pentapeptide that was originally isolated as a fungal natural product. It competitively inhibits family 18 chitinases by mimicking the chitooligosaccharide substrate of these enzymes. Interestingly, argifin is a nanomolar inhibitor of the bacterial-type subfamily of fungal chitinases that possess an extensive chitin-binding groove, but does not inhibit the much smaller, plant-type enzymes from the same family that are involved in fungal cell division and are thought to be potential drug targets. Here we show that a small, highly efficient, argifin-derived nine-atom fragment is a micromolar inhibitor of the plant-type chitinase ChiA1 from the opportunistic pathogen Aspergillus fumigatus. Evaluation of the binding mode with the first crystal structure of an A. fumigatus plant-type chitinase reveals that the compound binds the catalytic machinery in the same manner as observed for argifin with the bacterial-type chitinases. The structure of the complex was used to guide synthesis of derivatives to explore a pocket near the catalytic machinery. This work provides synthetically tractable plant-type family 18 chitinase inhibitors from the repurposing of a natural product.
Legionnaires’ disease is caused by a lethal colonization of alveolar macrophages with the Gram-negative bacterium Legionella pneumophila. LpGT (L. pneumophila glucosyltransferase; also known as Lgt1) has recently been identified as a virulence factor, shutting down protein synthesis in the human cell by specific glucosylation of EF1A (elongation factor 1A), using an unknown mode of substrate recognition and a retaining mechanism for glycosyl transfer. We have determined the crystal structure of LpGT in complex with substrates, revealing a GT-A fold with two unusual protruding domains. Through structure-guided mutagenesis of LpGT, several residues essential for binding of the UDP-glucose-donor and EF1A-acceptor substrates were identified, which also affected L. pneumophila virulence as demonstrated by microinjection studies. Together, these results suggested that a positively charged EF1A loop binds to a negatively charged conserved groove on the LpGT structure, and that two asparagine residues are essential for catalysis. Furthermore, we showed that two further L. pneumophila glycosyltransferases possessed the conserved UDP-glucose-binding sites and EF1A-binding grooves, and are, like LpGT, translocated into the macrophage through the Icm/Dot (intracellular multiplication/defect in organelle trafficking) system.
elongation factor 1A (EF1A); glucosyl transferase; Legionella pneumophila; microinjection; site-directed mutagenesis; protein structure
Although the risk of hepatitis B virus (HBV) was reported to be higher in military personnel than the general population in Saudi Arabia (SA), there is lack of studies assessing HBV awareness among them. The objective was to evaluate the knowledge, attitude and practice (KAP) of HBV infection among military personnel.
An intervention design with pre- and post-education KAP questionnaire was completed among National Guard soldiers working in Jeddah during January 2009. Educational intervention was provided through educational leaflets, group and individual discussions, visual show, and a lecture. A score was created from the correct answers to 58 questions.
A total of 400 male soldiers with mean age 30.7 ± 6.1 years completed both questionnaires. The majority had school education (96.8%) and in the lower military ranks (66.0%). Only 19.5% of soldiers reported HBV vaccine intake. The low median and inter-quartile range of the pre-intervention score (16, 6–26) markedly increased after education (to 53, 50–55, p<0.001). The overall improvement of mean KAP score (204%) was also observed in all its component scores; disease nature (272%), methods of transmission (206%), prevention and control (109%), attitude (155%), and practice (192%). The improvement was evident irrespective of socio-demographic characteristics and history of HBV vaccine. KAP scores were significantly associated with higher educational levels, higher monthly income, administrative jobs, and higher job ranks.
We are reporting a low level of HBV awareness among Saudi military population. The study confirms the need and effectiveness of focused multifaceted educational campaigns among the military population.
Hepatitis B virus; Knowledge; Attitude and Practice; Military; Saudi Arabia
O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release
The protein kinase TAK1 plays an important role in pro-inflammatory cytokine signalling. Interleukin-1- and osmotic stress-induced O-GlcNAcylation of its regulatory subunit TAB1 is required for full TAK1 activation to induce downstream cytokine production, linking this protein modification to innate immunity signalling.
Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is a key serine/threonine protein kinase that mediates signals transduced by pro-inflammatory cytokines such as transforming growth factor-β, tumour necrosis factor (TNF), interleukin-1 (IL-1) and wnt family ligands. TAK1 is found in complex with binding partners TAB1–3, phosphorylation and ubiquitination of which has been found to regulate TAK1 activity. In this study, we show that TAB1 is modified with N-acetylglucosamine (O-GlcNAc) on a single site, Ser395. With the help of a novel O-GlcNAc site-specific antibody, we demonstrate that O-GlcNAcylation of TAB1 is induced by IL-1 and osmotic stress, known inducers of the TAK1 signalling cascade. By reintroducing wild-type or an O-GlcNAc-deficient mutant TAB1 (S395A) into Tab1−/− mouse embryonic fibroblasts, we determined that O-GlcNAcylation of TAB1 is required for full TAK1 activation upon stimulation with IL-1/osmotic stress, for downstream activation of nuclear factor κB and finally production of IL-6 and TNFα. This is one of the first examples of a single O-GlcNAc site on a signalling protein modulating a key innate immunity signalling pathway.
cytokine; glycobiology; innate immunity; O-GlcNAc; signal transduction
Vibrio cholerae is a bacterial pathogen that colonizes the chitinous exoskeleton of zooplankton as well as the human gastrointestinal tract. Colonization of these different niches involves an N-acetylglucosamine binding protein (GbpA) that has been reported to mediate bacterial attachment to both marine chitin and mammalian intestinal mucin through an unknown molecular mechanism. We report structural studies that reveal that GbpA possesses an unusual, elongated, four-domain structure, with domains 1 and 4 showing structural homology to chitin binding domains. A glycan screen revealed that GbpA binds to GlcNAc oligosaccharides. Structure-guided GbpA truncation mutants show that domains 1 and 4 of GbpA interact with chitin in vitro, whereas in vivo complementation studies reveal that domain 1 is also crucial for mucin binding and intestinal colonization. Bacterial binding studies show that domains 2 and 3 bind to the V. cholerae surface. Finally, mouse virulence assays show that only the first three domains of GbpA are required for colonization. These results explain how GbpA provides structural/functional modular interactions between V. cholerae, intestinal epithelium and chitinous exoskeletons.
Vibrio cholerae is the bacterium that causes cholera, a disease endemic in developing countries with poor sanitation. The bacterium colonizes aquatic organisms that serve as a reservoir of transmission to humans. Our work has focused on GbpA, a protein that is secreted by V. cholerae and appears to facilitate growth of the bacteria both in the human intestine and on the exoskeletons of marine organisms. We show that the protein possesses an unusual three-dimensional structure consisting of four separate domains. Two of the domains are similar to proteins that are known to bind chitin, an exoskeleton biopolymer, and our data show that these domains indeed harbour the chitin binding properties of GbpA. One of these domains is also capable of binding intestinal mucus. The two remaining domains are required for interacting with the bacterium itself, creating a stable interface between the bacterium and the human/marine host, facilitating colonization. Finally, work with a cholera mouse model shows that only the first three domains of GbpA are required for colonization. These results show how GbpA provides structural/functional modular interactions between V. cholerae, the intestinal epithelium and chitinous exoskeletons.
BACKGROUND AND OBJECTIVES:
Few studies have been conducted in Saudi Arabia to estimate the prevalence of visual impairment and its causes. The objective of this study was to estimate the prevalence of visual impairment, and identify its causes and associated factors among the adult population attending primary health care (PHC) centers in Aljouf province, in northern Saudi Arabia.
DESIGN AND SETTING:
A cross-sectional study during the year 2005 in PHC centers in Aljouf province in northern Saudi Arabia
PATIENTS AND METHODS:
A sample of 620 Saudi adults, of age 18 years and older, from the catchment area of the Aljouf PHC centers, were randomly selected through a multistage random sampling technique. Data were collected using a questionnaire about socioeconomic and related information and a visual acuity test was performed using the Snellen chart (E). Diagnosis was established according to World Health Organization (WHO) criteria. Visual impairment was categorized into blindness for a visual acuity of less than 3/60 (20/400, 0.05) in the better eye with the best correction and low vision for a best corrected visual acuity of less than 6/18 (20/60, 0.3) but not less than 3/60 (20/400, 0.05) in the better eye. Regression analysis was used to identify the predictors of visual impairment.
Of 617 adult Saudis interviewed and examined, 269 (43.6%) were females. The mean (SD) age was 38.6 (16.2) years. The overall prevalence of visual impairment was 13.9% (95% CI: 11.4%-16.9%). The main medical causes of visual impairments were refractive errors (36.0%) followed by cataract (29.1%) and diabetic retinopathy (20.9%), and the least leading cause was glaucoma (5.8%). The most prominent determinants of visual impairment were age (P<.05), sex (P<.001), and a history of previous eye injury (P<.05).
Prevalence of visual impairment in the study population from the Aljouf area is high. It is recommended that regular checks of visual acuity be conducted for all Saudis of age 50+ years, who attend the PHC centers.
Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.
glycobiology; O-GlcNAc; protein structure; signal transduction
The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.