Structural and Biochemical Insights into the Mechanism of Action of the Clinical USP1 Inhibitor, KSQ-4279.

DNA damage triggers cell signaling cascades that mediate repair. This signaling is frequently dysregulated in cancers. The proteins that mediate this signaling are potential targets for therapeutic intervention. Ubiquitin-specific protease 1 (USP1) is one such target, with small-molecule inhibitors already in clinical trials. Here, we use biochemical assays and cryo-electron microscopy (cryo-EM) to study the clinical USP1 inhibitor, KSQ-4279 (RO7623066), and compare this to the well-established tool compound, ML323. We find that KSQ-4279 binds to the same cryptic site of USP1 as ML323 but disrupts the protein structure in subtly different ways. Inhibitor binding drives a substantial increase in thermal stability of USP1, which may be mediated through the inhibitors filling a hydrophobic tunnel-like pocket in USP1. Our results contribute to the understanding of the mechanism of action of USP1 inhibitors at the molecular level.

Capturing the catalytic intermediates of parkin ubiquitination.

Parkin is an E3 ubiquitin ligase implicated in early-onset forms of Parkinson's disease. It catalyzes a transthiolation reaction by accepting ubiquitin (Ub) from an E2 conjugating enzyme, forming a short-lived thioester intermediate, and transfers Ub to mitochondrial membrane substrates to signal mitophagy. A major impediment to the development of Parkinsonism therapeutics is the lack of structural and mechanistic detail for the essential, short-lived transthiolation intermediate. It is not known how Ub is recognized by the catalytic Rcat domain in parkin that enables Ub transfer from an E2~Ub conjugate to the catalytic site and the structure of the transthiolation complex is undetermined. Here, we capture the catalytic intermediate for the Rcat domain of parkin in complex with ubiquitin (Rcat-Ub) and determine its structure using NMR-based chemical shift perturbation experiments. We show that a previously unidentified α-helical region near the Rcat domain is unmasked as a recognition motif for Ub and guides the C-terminus of Ub toward the parkin catalytic site. Further, we apply a combination of guided AlphaFold modeling, chemical cross-linking, and single turnover assays to establish and validate a model of full-length parkin in complex with UbcH7, its donor Ub, and phosphoubiquitin, trapped in the process of transthiolation. Identification of this catalytic intermediate and orientation of Ub with respect to the Rcat domain provides important structural insights into Ub transfer by this E3 ligase and explains how the previously enigmatic Parkinson's pathogenic mutation T415N alters parkin activity.

A mechanistic review of Parkin activation.

Parkin and phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) constitute a feed-forward signalling pathway that mediates autophagic removal of damaged mitochondria (mitophagy). With over 130 mutations identified to date in over 1000 patients with early onset parkinsonism, Parkin is considered a hot spot of signalling pathways involved in PD aetiology. Parkin is an E3 ligase and how its activity is regulated has been extensively studied: inter-domain interactions exert a tight inhibition on Parkin activity; binding to phospho-ubiquitin relieves this auto-inhibition; and phosphorylation of Parkin shifts the equilibrium towards maximal Parkin activation. This review focusses on recent, structural findings on the regulation of Parkin activity. What follows is a mechanistic introduction to the family of E3 ligases that includes Parkin, followed by a brief description of structural elements unique to Parkin that lock the enzyme in an autoinhibited state, contrasted with emerging models that have shed light on possible mechanisms of Parkin activation.

An intellectual disability syndrome with single-nucleotide variants in O-GlcNAc transferase.

Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with ß-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.

A missense mutation in the catalytic domain of O-GlcNAc transferase links perturbations in protein O-GlcNAcylation to X-linked intellectual disability.

X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is. Here, we report the discovery of a missense mutation in the catalytic domain of OGT in an XLID patient. X-ray crystallography reveals that this variant leads to structural rearrangements in the catalytic domain. The mutation reduces in vitro OGT activity on substrate peptides/protein. Mouse embryonic stem cells carrying the mutation reveal reduced O-GlcNAcase (OGA) and global O-GlcNAc levels. These data suggest a direct link between changes in the O-GlcNAcome and intellectual disability observed in patients carrying OGT mutations.

Catalytic deficiency of O-GlcNAc transferase leads to X-linked intellectual disability.

O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.

Structural basis of generic versus specific E2-RING E3 interactions in protein ubiquitination.

Protein ubiquitination is a fundamental regulatory component in eukaryotic cell biology, where a cascade of ubiquitin activating (E1), conjugating (E2), and ligating (E3) enzymes assemble distinct ubiquitin signals on target proteins. E2s specify the type of ubiquitin signal generated, while E3s associate with the E2~Ub conjugate and select the substrate for ubiquitination. Thus, producing the right ubiquitin signal on the right target requires the right E2-E3 pair. The question of how over 600 E3s evolved to discriminate between 38 structurally related E2s has therefore been an area of intensive research, and with over 50 E2-E3 complex structures generated to date, the answer is beginning to emerge. The following review discusses the structural basis of generic E2-RING E3 interactions, contrasted with emerging themes that reveal how specificity can be achieved.

The O-GlcNAc Transferase Intellectual Disability Mutation L254F Distorts the TPR Helix.

O-linked ß-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis.

Mutations in N-acetylglucosamine (O-GlcNAc) transferase in patients with X-linked intellectual disability.

N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.

Evidence for a Functional O-Linked N-Acetylglucosamine (O-GlcNAc) System in the Thermophilic Bacterium Thermobaculum terrenum.

Post-translational modification of proteins is a ubiquitous mechanism of signal transduction in all kingdoms of life. One such modification is addition of O-linked N-acetylglucosamine to serine or threonine residues, known as O-GlcNAcylation. This unusual type of glycosylation is thought to be restricted to nucleocytoplasmic proteins of eukaryotes and is mediated by a pair of O-GlcNAc-transferase and O-GlcNAc hydrolase enzymes operating on a large number of substrate proteins. Protein O-GlcNAcylation is responsive to glucose and flux through the hexosamine biosynthetic pathway. Thus, a close relationship is thought to exist between the level of O-GlcNAc proteins within and the general metabolic state of the cell. Although isolated apparent orthologues of these enzymes are present in bacterial genomes, their biological functions remain largely unexplored. It is possible that understanding the function of these proteins will allow development of reductionist models to uncover the principles of O-GlcNAc signaling. Here, we identify orthologues of both O-GlcNAc cycling enzymes in the genome of the thermophilic eubacterium Thermobaculum terrenum. The O-GlcNAcase and O-GlcNAc-transferase are co-expressed and, like their mammalian orthologues, localize to the cytoplasm. The O-GlcNAcase orthologue possesses activity against O-GlcNAc proteins and model substrates. We describe crystal structures of both enzymes, including an O-GlcNAcase·peptide complex, showing conservation of active sites with the human orthologues. Although in vitro activity of the O-GlcNAc-transferase could not be detected, treatment of T. terrenum with an O-GlcNAc-transferase inhibitor led to inhibition of growth. T. terrenum may be the first example of a bacterium possessing a functional O-GlcNAc system.

Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors.

Inhibitors of OGT (O-GlcNAc transferase) are valuable tools to study the cell biology of protein O-GlcNAcylation. We report OGT bisubstrate-linked inhibitors (goblins) in which the acceptor serine in the peptide VTPVSTA is covalently linked to UDP, eliminating the GlcNAc pyranoside ring. Goblin1 co-crystallizes with OGT, revealing an ordered C3 linker and retained substrate-binding modes, and binds the enzyme with micromolar affinity, inhibiting glycosyltransfer on to protein and peptide substrates.

Gender differences in severity of sickle cell diseases in non-smokers.

OBJECTIVE: To find out gender differences in severity of sickle cell diseases (SCDs) in non-smokers. METHODS: Three groups of SCDs patients on the basis of red blood cell (RBC) transfusions were included. Less than 10 units in their lives were kept in Group-1, Ten units of higher in Group-2 and 50 units or higher as the Third Group. Patients with a history of using one pack of cigarettes -year or above were excluded. RESULTS: The study included 269 patients. Mean ages of the groups were similar (28.4, 28.5, and 28.9 years, respectively). Prevalences of cases without any RBC transfusion in their lives were 7.2% and 3.7% in females and males, respectively (p<0.05). Prevalences of cases without any painful crisis were 13.8% and 6.0% in females and males, respectively (p<0.001). There was progressive increase according to mean painful crises, clubbing, chronic obstructive pulmonary disease (COPD), leg ulcers, stroke, chronic renal disease (CRD), pulmonary hypertension, and male ratio from the first towards the third groups (p<0.05, nearly for all). Mean ages of mortal cases were 29.1 and 26.2 years in females and males, respectively (p>0.05). CONCLUSION: The higher painful crises per year, digital clubbing, COPD, leg ulcers, stroke, CRD, pulmonary hypertension, and male ratio of the third group, lower male ratio of patients without any RBC transfusion, lower male ratio of patients without any painful crisis, lower mean ages of male SCDs patients with mortality, and longer overall survival of females in the world could not be explained by well known strong atherosclerotic effects of smoking alone, instead it may be explained by the dominant role of male sex in life.

Gender differences in patients with metabolic syndrome in coronary artery interventions.

AIM: We tried to understand whether or not there was a gender difference in coronary artery interventions in coronary heart disease (CHD) cases in the present study. METHODS: The study was performed in two phases. The first phase was performed at the Internal Medicine Polyclinic of the Dumlupinar University between August 2005 and March 2007. CHD was diagnosed either angiographically or with history of coronary artery stenting (CAS) and/or coronary artery bypass graft (CABG) surgery. The second phase was performed at the Internal Medicine Polyclinic of the Mustafa Kemal University between March 2007 and April 2012. During the second phase, the CHD patients with CAS and/or CABG surgery were detected and divided into two groups according to the gender. RESULTS: Mean age and prevalence of CHD were similar in both genders (p>0.05 for both) in the first phase. Smoking was higher in males with CHD, in 30 cases (54.5%) of males versus six (9.6%) cases of females (p < 0.001), as well as chronic obstructive pulmonary disease (COPD), in ten (18.1%) cases of males versus four (6.4%) cases of females (p < 0.05). Although the body mass index (BMI) and white coat hypertension (WCH) were insignificantly higher (p>0.05 for both), low density lipoprotein cholesterol (LDL-C) and triglyceride (TG) were significantly higher in females with CHD (p= 0.008 and p= 0.002, respectively). Hypertension (HT) and diabetes mellitus (DM) were higher in females with CHD, too (p < 0.001 and p < 0.05, respectively). On the other hand, CAS and/or CABG surgery were significantly higher in male CHD cases (21.8% versus 1.6%, p less 0.001). Parallel to the first phase cases, majority of CAS and/or CABG surgery cases were males in the second phase cases too (90.2% versus 9.7%, p less 0.001). CONCLUSION: As some components of the metabolic syndrome, smoking and COPD were higher in males whereas BMI, WCH, LDL-C, TG, HT and DM were higher in females. Despite similar prevalences of CHD in both sexes, CAS and/or CABG surgery were significantly higher in males probably due to fear of loss of power required for their dominant roles in life and sexuality.

Low-dose cytosine arabinoside-induced syptomatic bradycardia in a patient with acute myeloid leukemia.

Cytosine arabinoside (Ara-C) is one of the critical agents for the treatment of acute myeloid leukemia (AML). The toxicity profile of Ara-C is highly dependent on the dose and schedule of administration. Cardiologic complications associated with Ara-C are rare. These side effects were reported with high doses of cytarabine (1-3 g/m2, 6-12 doses) in the literature. Herein, we report a patient who developed symptomatic sinus bradycardia while receiving low-dose Ara-C therapy for AML. A 45-year-old female patient diagnosed with AML was treated with standard remission induction chemotherapy protocol that includes 3 days of anthracycline and 7 days of low-dose (100-200 mg/m2 2-1) Ara-C. The same chemotherapy regimen was applied again on the 15th day of admission. During the second chemotherapy cycle, the patient developed symptomatic sinus bradycardia. All causes except Ara-C were excluded after required investigational procedures. Ara-C infusion was discontinued for a while and after her symptoms passed chemotherapy was completed with atropine support. Cardiac toxicity is scarce with Ara-C. We want to remind that clinicians should be aware of this potential toxic manifestation even in low doses of the medication, especially as Ara-C is widely used in the treatment of leukemia. .

Acute promyelocytic leukemia, centre, experience, Turkey.

Acute promyelocytic leukemia (APL) is a specific type of acute myeloid leukemia (AML) and has distinct hematopathologic, cytogenetic, clinical and molecular features. This study was a retrospective review of 18 adult patients (10 male, 8 female; mean age of 32.17 ± 5.66 (15-61 years) with APL at our department from January 2006 to December 2011. Following induction therapy, 17 patients achieved CR, 1 of 18 patients died of result bleeding within thirty-sixth hours of admission. In two of 18 patients developed RAS. The relapse rate was 27% (5/18). Fourteen of 18 patients (77%) have been followed in remission. APL is a malignancy requiring quick diagnosis, efficient treatment and supportive care system. ATO, one of the important therapy option in the treatment of APL, cannot be obtained easily in developing countries. This may lead to an increase in the mortality rates. The studies should be made with more number of patients and a longer period of time for accurate results.

White coat hypertension may be an initial sign of the metabolic syndrome.

AIM: to understand the role and significance of WCH in definition of the metabolic syndrome. METHODS: the study was performed in the Internal Medicine Polyclinic of the Dumlupinar University between August 2005 and March 2007. We took consecutive patients at and above the age of 20 years. Their medical histories including smoking habit, DM, dyslipidemia, and already used medications were learnt, and a routine check up procedure including fasting plasma glucose (FPG), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), and an electrocardiography was performed. Comparison of proportions was used as the method of statistical analysis. RESULTS: the study included 1,068 cases. Prevalences of excess weight increased from the third (28.7%) up to the seventh decades (87.0%), gradually (p<0.05 nearly in all steps), and then decreased in the eighth (78.5%, p<0.05) decade of life. The most significant increase was seen during the passage from the third to the fourth decades (28.7% versus 63.6%, p<0.001) with a similar fashion to smoking. Hyperbetalipoproteinemia, hypertriglyceridemia, dyslipidemia, impaired glucose tolerance (IGT), and WCH had a similar fashion with excess weight by increasing until the seventh decade and decreasing afterwards (p<0.05 nearly in all steps). Whereas hypertension (HT), type 2 diabetes mellitus (DM), and coronary heart disease (CHD) always increased without any decrease by decades (p<0.05 nearly in all steps), indicating their irreversible natures. CONCLUSION: WCH may be an initial sign of the systemic atherosclerotic process that can be detected easily and prevented by a trend towards weight loss.

Mercury toxicity: a family case report.

BACKGROUND: Environmental pollution exposes humans to toxic substances. Herein we present 5 family members aged20-54 years that were poisoned by liquid mercury. CASE REPORTS: Case 1 presented to our clinic with cough, fever, and night sweats. The patient had neutropenia, anemia,and pneumonia, rapidly developed acute respiratory distress syndrome (ARDS), and died on day 4 of hospitalization.Her WBC count was 0.4 × 10³ mm-3 (normal range: 4.3-10.3 × 103 mm-3) and Hb was 10.8 g dL-1 (normal range: 11.5-16.0 g dL-1). Case 2 presented with bicytopenia; the leukocyte count was 1.3 × 103 mm-3 (normal range: 4.3-10.3 × 103mm-3) and the PLT count was 88 × 103 mm-3 (normal range: 150-400 × 103 mm-3). Cases 2 and 3 had toxic peripheralneuropathy. The PLT count in case 3 was 123 × 103 mm-3 (normal range: 150-400 × 103 mm-3). Cases 4 and 5 presentedwith fatigue and headache; these 2 patients did not have positive findings, apart from high levels of mercury in theblood. We have written informed consent. CONCLUSION: We think that heavy metal exposure-although rare-should be considered in patients that present withnumerous symptoms involving multiple systems, including the cardiovascular, respiratory, and neurological systems.The present report is unique in that in describes mercury poisoning in 5 members of the same family.

Thrombotic thrombocytopenic purpura: A single - Centre experience.

We aimed to describe the characteristics, treatment regime, and 6-month all-cause mortality of thrombotic thrombocytopenic purpura (TTP) patients treated with total plasma exchange in the our clinic. Thirteen patients were included in the study. Mortality rates of TTP have improved over the last three decades but they are still too high according to modern therapy expectations. Etiology directed treatment should be added to total plasma exchange in secondary TTP cases. Based on TTPs' immunologic etiology, immune modulator and immune suppressor agents have been applied together with total plasma exchange, but mostly in anecdotal case reports or with questionable responses.

The Levels of Serum C-Reactive Protein, Beta 2 Microglobulin, Ferritin, Lactate Dehydrogenase and Some Specific Proteins in Patients with Non-Hodgkin's Lymphoma Before and After Treatment.

OBJECTIVE: The aim of this study was to measure serum C reactive protein, ß2 microglobulin, ferritin, lactate dehydrogenase, complement 3, complement 4, immunoglobulin A, immunoglobulin M, immunoglobulin G and transferrin levels in patients with Non-Hodgkin Lymphoma before and after treatment, and to determine whether any differences occur with treatment, investigate relationship between these parameters and systemic symptoms, and to determine whether they could be used as tumor markers. MATERIALS AND METHODS: The parameters listed above were studied before and after treatment in sera of 27 patients with the diagnosis of Non-Hodgkin Lymphoma who admitted to our department. Of the patients, 10 (37%) were females and 17 (63%) were males. Mean age was 57.7 ± 16.5 (19-82) years. The subjects were newly diagnosed and treatment. RESULTS: Post-treatment serum ferritin and CRP levels were found to be significantly decreased in patients with NHL compared to pre-treatment levels (p=0.009 and p=0.015, respectively). In addition, ferritin levels measured before treatment were significantly lower in subjects with B symptoms than those without B symptoms (p=0.02). IgA levels of patients with B symptom were significantly increased compared to those without B symptoms following treatment (p=0.03). CONCLUSIONS: We are in the opinion that serum ferritin and CRP parameters may be used as tumor markers and may be indicators in the efficacy evaluation of treatment in Non-Hodgkin's Lymphoma.

A simple, safe and effective approach to prevent postdural puncture headache: epidural saline injection.

OBJECTIVE: In this study, we investigated the safety and effectiveness of epidural saline injection to prevent post-dural puncture headache (PDPH) in patients with acute lymphoblastic leukemia (ALL). MATERIALS AND METHODS: Thirty-three patients with ALL undergoing induction therapy were accepted for the study. Four to six courses of intrathecal methotrexate therapy were administered to each patient for central nervous system prophylaxis. Patients were divided into two groups. Lumbar puncture (LP) was performed without any additional intervention in the first group (18 cases), whereas 20 mL of isotonic saline was injected into the epidural space in the second group (15 cases). The frequency and severity of PDPH were compared between the two groups. RESULTS: Thirteen patients from the first group and five patients from the second group experienced at least one PDPH episode. In total, 54 PDPH episodes were reported in both groups. The rate of headache due to the LP was significantly higher in the first group than in the second group (48.8% vs. 16.4%, p<0.001). On the other hand, the severity of pain was also significantly higher in the first group (mean pain scores were 5.6 ± 1.62 vs. 3.07 ± 1.18, p<0.001). Furthermore, two patients from the first group (11.11%) developed generalized convulsion attacks, and one of those patients experienced pulmonary arrest necessitating respiratory support. No serious complications were observed in the second group. CONCLUSIONS: Our study shows that isotonic saline injection into the epidural space after LP is a safe and effective approach to prevent PDPH and related complications.