Ria Weston

The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase

Post-translational modification of proteins by poly(ADP-ribosyl)ation regulates many cellular pathways that are critical for genome stability, including DNA repair, chromatin structure, mitosis and apoptosis. Poly(ADP-ribose) (PAR) is composed of repeating ADP-ribose units linked via a unique glycosidic ribose–ribose bond, and is synthesized from NAD by PAR polymerases. PAR glycohydrolase (PARG) is the only protein capable of specific hydrolysis of the ribose–ribose bonds present in PAR chains; its deficiency leads to cell death. Here we show that filamentous fungi and a number of bacteria possess a divergent form of PARG that has all the main characteristics of the human PARG enzyme. We present the first PARG crystal structure (derived from the bacterium Thermomonospora curvata), which reveals that the PARG catalytic domain is a distant member of the ubiquitous ADP-ribose-binding macrodomain family. High-resolution structures of T. curvata PARG in complexes with ADP-ribose and the PARG inhibitor ADP-HPD, complemented by biochemical studies, allow us to propose a model for PAR binding and catalysis by PARG. The insights into the PARG structure and catalytic mechanism should greatly improve our understanding of how PARG activity controls reversible protein poly(ADP-ribosyl)ation and potentially of how the defects in this regulation are linked to human disease.

Deficiency of terminal ADP‐ribose protein glycohydrolase TARG1/C6orf130 in neurodegenerative disease

Adenosine diphosphate (ADP)‐ribosylation is a post‐translational protein modification implicated in the regulation of a range of cellular processes. A family of proteins that catalyse ADP‐ribosylation reactions are the poly(ADP‐ribose) (PAR) polymerases (PARPs). PARPs covalently attach an ADP‐ribose nucleotide to target proteins and some PARP family members can subsequently add additional ADP‐ribose units to generate a PAR chain. The hydrolysis of PAR chains is catalysed by PAR glycohydrolase (PARG). PARG is unable to cleave the mono(ADP‐ribose) unit directly linked to the protein and although the enzymatic activity that catalyses this reaction has been detected in mammalian cell extracts, the protein(s) responsible remain unknown. Here, we report the homozygous mutation of the c6orf130 gene in patients with severe neurodegeneration, and identify C6orf130 as a PARP‐interacting protein that removes mono(ADP‐ribosyl)ation on glutamate amino acid residues in PARP‐modified proteins. X‐ray structures and biochemical analysis of C6orf130 suggest a mechanism of catalytic reversal involving a transient C6orf130 lysyl‐(ADP‐ribose) intermediate. Furthermore, depletion of C6orf130 protein in cells leads to proliferation and DNA repair defects. Collectively, our data suggest that C6orf130 enzymatic activity has a role in the turnover and recycling of protein ADP‐ribosylation, and we have implicated the importance of this protein in supporting normal cellular function in humans.

DNA Repair Factor APLF Is a Histone Chaperone

Poly(ADP-ribosyl)ation plays a major role in DNA repair, where it regulates chromatin relaxation as one of the critical events in the repair process. However, the molecular mechanism by which poly(ADP-ribose) modulates chromatin remains poorly understood. Here we identify the poly(ADP-ribose)-regulated protein APLF as a DNA-damage-specific histone chaperone. APLF preferentially binds to the histone H3/H4 tetramer via its C-terminal acidic motif, which is homologous to the motif conserved in the histone chaperones of the NAP1L family (NAP1L motif). We further demonstrate that APLF exhibits histone chaperone activities in a manner that is dependent on its acidic domain and that the NAP1L motif is critical for the repair capacity of APLF in vivo. Finally, we identify structural analogs of APLF in lower eukaryotes with the ability to bind histones and localize to the sites of DNA-damage-induced poly(ADP-ribosyl)ation. Collectively, these findings define the involvement of histone chaperones in poly(ADP-ribose)-regulated DNA repair reactions.

Endothelial microparticles as conveyors of information in atherosclerotic disease

Endothelial microparticles (EMPs) are complex submicron membrane-shed vesicles released into the circulation following endothelium cell activation or apoptosis. They are classified as either physiological or pathological, with anticoagulant or pro-inflammatory effects respectively. Endothelial dysfunction caused by inflammation is a key initiating event in atherosclerotic plaque formation. Athero-emboli, resulting from ruptured carotid plaques are a major cause of stroke. Current clinical techniques for arterial assessment, angiography and carotid ultrasound, give accurate information about stenosis but limited evidence on plaque composition, inflammation or vulnerability; as a result, patients with asymptomatic, or fragile carotid lesions, may not be identified and treated effectively. There is a need to discover novel biomarkers and develop more efficient diagnostic approaches in order to stratify patients at most risk of stroke, who would benefit from interventional surgery. Increasing evidence suggests that EMPs play an important role in the pathogenesis of cardiovascular disease, acting as a marker of damage, either exacerbating disease progression or triggering a repair response. In this regard, it has been suggested that EMPs have the potential to act as biomarkers of disease status. In this review, we will present the evidence to support this hypothesis and propose a novel concept for the development of a diagnostic device that could be implemented in the clinic.

Monomeric C-reactive protein-a key molecule driving development of Alzheimer’s disease associated with brain ischaemia?

Alzheimer’s disease (AD) increases dramatically in patients with ischaemic stroke. Monomeric C-reactive protein (mCRP) appears in the ECM of ischaemic tissue after stroke, associating with microvasculature, neurons and AD-plaques, Aβ, also, being able to dissociate native-CRP into inflammatory, mCRP in vivo. Here, mCRP injected into the hippocampal region of mice was retained within the retrosplenial tract of the dorsal 3rd ventrical and surrounding major vessels. Mice developed behavioural/cognitive deficits within 1 month, concomitant with mCRP staining within abnormal looking neurons expressing p-tau and in beta-amyloid 1-42-plaque positive regions. mCRP co-localised with CD105 in microvessels suggesting angiogenesis. Phospho-arrays/Western blotting identified signalling activation in endothelial cells and neurons through p-IRS-1, p-Tau and p-ERK1/2-which was blocked following pre-incubation with mCRP-antibody. mCRP increased vascular monolayer permeability and gap junctions, increased NCAM expression and produced haemorrhagic angiogenesis in mouse matrigel implants. mCRP induced tau244–372 aggregation and assembly in vitro. IHC study of human AD/stroke patients revealed co-localization of mCRP with Aβ plaques, tau-like fibrils and IRS-1/P-Tau positive neurons and high mCRP-levels spreading from infarcted core regions matched reduced expression of Aβ/Tau. mCRP may be responsible for promoting dementia after ischaemia and mCRP clearance could inform therapeutic avenues to reduce the risk of future dementia.

Elevated levels of endothelial-derived microparticles, and serum CXCL9 and SCGF-β are associated with unstable asymptomatic carotid plaques.

Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques, and may correlate with serum markers of plaque instability and inflammation. Circulating EMPs, platelet MPs (PMPs) and inflammatory markers were measured in healthy controls and patients undergoing carotid endarterectomy. EMP/PMPs were quantified using flow cytometry. Bioplex assays profiled systemic inflammatory and bone-related proteins. Immunohistological analysis detailed the contribution of differentially-regulated systemic markers to plaque pathology. Alizarin red staining showed calcification. EMPs and PMPs were significantly higher in patients with carotid stenosis (≥70%) compared to controls, with no differences between asymptomatic vs symptomatic patients. Asymptomatic patients with unstable plaques exhibited higher levels of EMPs, CXCL9 and SCGF-β compared to those with stable plaques. CXCL9, and SCGF-β were detected within all plaques, suggesting a contribution to both localised and systemic inflammation. Osteopontin and osteoprotegerin were significantly elevated in the symptomatic vs asymptomatic group, while osteocalcin was higher in asymptomatic patients with stable plaque. All plaques exhibited calcification, which was significantly greater in asymptomatic patients. This may impact on plaque stability. These data could be important in identifying patients at most benefit from intervention.

Up-regulation of serotonin receptor 2B mRNA and protein in the peri-infarcted area of aged rats and stroke patients

Despite the fact that a high proportion of elderly stroke patients develop mood disorders, the mechanisms underlying late-onset neuropsychiatric and neurocognitive symptoms have so far received little attention in the field of neurobiology. In rodents, aged animals display depressive symptoms following stroke, whereas young animals recover fairly well. This finding has prompted us to investigate the expression of serotonin receptors 2A and 2B, which are directly linked to depression, in the brains of aged and young rats following stroke. Although the development of the infarct was more rapid in aged rats in the first 3 days after stroke, by day 14 the cortical infarcts were similar in size in both age groups i.e. 45% of total cortical volume in young rats and 55.7% in aged rats. We also found that the expression of serotonin receptor type B mRNA was markedly increased in the perilesional area of aged rats as compared to the younger counterparts. Furthermore, histologically, HTR2B protein expression in degenerating neurons was closely associated with activated microglia both in aged rats and human subjects. Treatment with fluoxetine attenuated the expression of Htr2B mRNA, stimulated post-stroke neurogenesis in the subventricular zone and was associated with an improved anhedonic behavior and an increased activity in the forced swim test in aged animals. We hypothesize that HTR2B expression in the infarcted territory may render degenerating neurons susceptible to attack by activated microglia and thus aggravate the consequences of stroke.

HIV-1 matrix protein p17 misfolding forms toxic amyloidogenic assemblies that induce neurocognitive disorders

Human immunodeficiency virus type-1 (HIV-1)-associated neurocognitive disorder (HAND) remains an important neurological manifestation that adversely affects a patient’s quality of life. HIV-1 matrix protein p17 (p17) has been detected in autoptic brain tissue of HAND individuals who presented early with severe AIDS encephalopathy. We hypothesised that the ability of p17 to misfold may result in the generation of toxic assemblies in the brain and may be relevant for HAND pathogenesis. A multidisciplinary integrated approach has been applied to determine the ability of p17 to form soluble amyloidogenic assemblies in vitro. To provide new information into the potential pathogenic role of soluble p17 species in HAND, their toxicological capability was evaluated in vivo. In C. elegans, capable of recognising toxic assemblies of amyloidogenic proteins, p17 induces a specific toxic effect which can be counteracted by tetracyclines, drugs able to hinder the formation of large oligomers and consequently amyloid fibrils. The intrahippocampal injection of p17 in mice reduces their cognitive function and induces behavioral deficiencies. These findings offer a new way of thinking about the possible cause of neurodegeneration in HIV-1-seropositive patients, which engages the ability of p17 to form soluble toxic assemblies.

179 The Role Of Microparticles in Carotid Disease

Introduction Endothelial microparticles (EMPs) are released from dysfunctional endothelial cells. We hypothesised that patients with unstable carotid plaque have higher levels of circulating microparticles compared to patients with stable plaques which could be related to a specific cytokine profile. Methods Circulating EMPs and inflammatory cytokine levels were measured in seventy patients with significant carotid disease undergoing carotid endarterectomy and 20 healthy controls. Fifty one (73%) patients had symptomatic disease whilst 19 (27%) were asymptomatic. EMPs (CD31+/ Annexin V+ CD42b-) were quantified using flow cytometry. Immunohistological analysis of carotid plaques for CD68+, CD206+ macrophages, TNF-α smooth muscle actin and osteopontin was performed, together with Alizarin red staining for detection of calcific deposits. Bioplex assays were used for cytokine analysis. Plaques were graded according to the American Heart Association plaque scoring system. Results Significantly higher EMP levels were observed in symptomatic patients compared to controls, p = 0.01, while no differences were noted in EMP levels in asymptomatic vs controls p = 0.11. The higher EMP levels appeared to associate with the unstable plaques which also had a significantly higher level of CD68+ macrophages compared to stable plaques (AHA I-IV) and higher circulating levels of Chemokine ligand-9 (CXCL-9) (p < 0.004). Of note, macrophage inhibitory factor (MIF) was among the chemokines that were elevated in the circulation of patients with stable plaques, whether a lack of this factor in unstable plaques has direct effects on phenotype remains to be elucidated. Other factors elevated in patients with stable plaques were IL-16, cutaneous T-cell attracting chemokine (CTACK), and stem-cell growth factor-b (SCGF-b) (p value of 0.05, 0.035 and 0.002 respectively). Conclusion Circulatory EMP and specific inflammatory cytokine levels are raised in patients with unstable plaques, while MIF, a potentially protective factor was elevated in serum of patients with stable plaques. These data could have major implications for the development of a diagnostic tool whereby EMPs together with markers of macrophage activity could act in a combined manner as biomarkers of plaque vulnerability and stroke susceptibility.

183 Novel glycomimetics inhibit gly-ldl induced smooth muscle cell calcification via creb

Advanced glycated end-products (AGEs) are known drivers of cardiovascular complications such as vascular calcification, which is currently untreatable, and are increased in diabetic subjects in part due to oxidative stress and poor glycaemic control. Our previous studies using smooth muscle cells (SMCs) isolated from patients with peripheral arterial disease (PAD) have implicated a number of signalling pathways involved in their osteogenic differentiation in vitro, including OPG/RANK. We have also shown that alterations in the cell surface glycocalyx regulates cell function, suggesting that non-sugar glycosaminoglycan mimics can potentially modulate cell phenotypes. We aim to investigate how modified LDL and PAD serum affects the progression of calcification of SMCs in vitro and whether this pathology can be prevented by novel glycosaminoglycan mimics, using qPCR, ELISA, Alkaline phosphatase (ALP) activity, and Western blotting. Gly-LDL (10 µg/ml) increased an early marker of calcification (ALP activity) at 4 days and enhanced calcification at 21 days, compared to controls (p<0.05) as shown by alizarin red staining. PAD serum accelerated calcification, which was apparent after 10 days (p<0.05). The glycomimetics significantly inhibited both gly-LDL and PAD serum-induced mineralisation, and reduced gly-LDL induced ALP activity at day 4 (p<0.05). In the gly-LDL-treated SMCs, secreted levels of osteocalcin (OCN), a promoter of osteogenic differentiation, were reduced when treated with the glycomimetics, whereas osteopontin (OPN) and osteoprotegerin (OPG), inhibitors of calcification, were increased compared to gly-LDL alone. A similar trend was observed in PAD serum-treated SMCs, with glycomimetics reducing OCN and increasing OPN secretion. A phospho-kinase array analysis of gly-LDL-treated SMCs was performed to identify underlying mechanism of action. Gly-LDL increased phosphorylation of cyclic AMP response element-binding protein (CREB), TOR, and the SRC proteins LYN, YES and CHK-2 compared with untreated control, which was attenuated with the addition of the glycomimetics. A number of upstream activators of CREB were targeted using known pharmacological inhibitors in SMCs treated with gly-LDL. The MEK inhibitor U0126 accelerated calcification, increasing both ALP activity and expression of receptor for AGEs (RAGE), a key receptor implicated in vascular calcification, compared to gly-LDL alone, suggesting that MEK may be a mediator of mineralisation process via phosphorylation of CREB. Glycomimetics have potential as an anti-calcification strategy, inhibiting mineralisation in SMCs induced by both gly-LDL and patient serum in vitro. The protective effect of glycomimetics against calcification may occur via regulation of CREB phosphorylation and subsequent modulation of downstream osteogenic markers, including upregulation of OPN and OPG and reduction of OCN, leading to the development of therapeutics to treat vascular calcification.