Constitutive phosphorylation of MDC1 physically links the MRE11–RAD50–NBS1 complex to damaged chromatin

The MRE11–RAD50–Nijmegen breakage syndrome 1 (NBS1 [MRN]) complex accumulates at sites of DNA double-strand breaks (DSBs) in microscopically discernible nuclear foci. Focus formation by the MRN complex is dependent on MDC1, a large nuclear protein that directly interacts with phosphorylated H2AX. In this study, we identified a region in MDC1 that is essential for the focal accumulation of the MRN complex at sites of DNA damage. This region contains multiple conserved acidic sequence motifs that are constitutively phosphorylated in vivo. We show that these motifs are efficiently phosphorylated by caseine kinase 2 (CK2) in vitro and directly interact with the N-terminal forkhead-associated domain of NBS1 in a phosphorylation-dependent manner. Mutation of these conserved motifs in MDC1 or depletion of CK2 by small interfering RNA disrupts the interaction between MDC1 and NBS1 and abrogates accumulation of the MRN complex at sites of DNA DSBs in vivo. Thus, our data reveal the mechanism by which MDC1 physically couples the MRN complex to damaged chromatin

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damag

Specific immune modulation of experimental colitis drives enteric alpha-synuclein accumulation and triggers age-related Parkinson-like brain pathology

Background: In some people with Parkinson’s disease (PD), a-synuclein (αSyn) accumulation may begin in the enteric nervous system (ENS) decades before development of brain pathology and disease diagnosis. Objective: To determine how different types and severity of intestinal inflammation could trigger αSyn accumulation in the ENS and the subsequent development of αSyn brain pathology. Methods: We assessed the effects of modulating short- and long-term experimental colitis on αSyn accumulation in the gut of αSyn transgenic and wild type mice by immunostaining and gene expression analysis. To determine the long-term effect on the brain, we induced dextran sulfate sodium (DSS) colitis in young αSyn transgenic mice and aged them under normal conditions up to 9 or 21 months before tissue analyses. Results: A single strong or sustained mild DSS colitis triggered αSyn accumulation in the submucosal plexus of wild type and αSyn transgenic mice, while short-term mild DSS colitis or inflammation induced by lipopolysaccharide did not have such an effect. Genetic and pharmacological modulation of macrophage-associated pathways modulated the severity of enteric αSyn. Remarkably, experimental colitis at three months of age exacerbated the accumulation of aggregated phospho-Serine 129 αSyn in the midbrain (including the substantia nigra), in 21- but not 9-month-old αSyn transgenic mice. This increase in midbrain αSyn accumulation is accompanied by the loss of tyrosine hydroxylase-immunoreactive nigral neurons. Conclusions: Our data suggest that specific types and severity of intestinal inflammation, mediated by monocyte/macrophage signaling, could play a critical role in the initiation and progression of PD

The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator

Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a ‘head-to-tail’ dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage

Development and Validation of a Risk Score for Chronic Kidney Disease in HIV Infection Using Prospective Cohort Data from the D:A:D Study

Ristola M. on työryhmien DAD Study Grp ; Royal Free Hosp Clin Cohort ; INSIGHT Study Grp ; SMART Study Grp ; ESPRIT Study Grp jäsen.Background Chronic kidney disease (CKD) is a major health issue for HIV-positive individuals, associated with increased morbidity and mortality. Development and implementation of a risk score model for CKD would allow comparison of the risks and benefits of adding potentially nephrotoxic antiretrovirals to a treatment regimen and would identify those at greatest risk of CKD. The aims of this study were to develop a simple, externally validated, and widely applicable long-term risk score model for CKD in HIV-positive individuals that can guide decision making in clinical practice. Methods and Findings A total of 17,954 HIV-positive individuals from the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study with >= 3 estimated glomerular filtration rate (eGFR) values after 1 January 2004 were included. Baseline was defined as the first eGFR > 60 ml/min/1.73 m2 after 1 January 2004; individuals with exposure to tenofovir, atazanavir, atazanavir/ritonavir, lopinavir/ritonavir, other boosted protease inhibitors before baseline were excluded. CKD was defined as confirmed (>3 mo apart) eGFR In the D:A:D study, 641 individuals developed CKD during 103,185 person-years of follow-up (PYFU; incidence 6.2/1,000 PYFU, 95% CI 5.7-6.7; median follow-up 6.1 y, range 0.3-9.1 y). Older age, intravenous drug use, hepatitis C coinfection, lower baseline eGFR, female gender, lower CD4 count nadir, hypertension, diabetes, and cardiovascular disease (CVD) predicted CKD. The adjusted incidence rate ratios of these nine categorical variables were scaled and summed to create the risk score. The median risk score at baseline was -2 (interquartile range -4 to 2). There was a 1: 393 chance of developing CKD in the next 5 y in the low risk group (risk score = 5, 505 events), respectively. Number needed to harm (NNTH) at 5 y when starting unboosted atazanavir or lopinavir/ritonavir among those with a low risk score was 1,702 (95% CI 1,166-3,367); NNTH was 202 (95% CI 159-278) and 21 (95% CI 19-23), respectively, for those with a medium and high risk score. NNTH was 739 (95% CI 506-1462), 88 (95% CI 69-121), and 9 (95% CI 8-10) for those with a low, medium, and high risk score, respectively, starting tenofovir, atazanavir/ritonavir, or another boosted protease inhibitor. The Royal Free Hospital Clinic Cohort included 2,548 individuals, of whom 94 individuals developed CKD (3.7%) during 18,376 PYFU (median follow-up 7.4 y, range 0.3-12.7 y). Of 2,013 individuals included from the SMART/ESPRIT control arms, 32 individuals developed CKD (1.6%) during 8,452 PYFU (median follow-up 4.1 y, range 0.6-8.1 y). External validation showed that the risk score predicted well in these cohorts. Limitations of this study included limited data on race and no information on proteinuria. Conclusions Both traditional and HIV-related risk factors were predictive of CKD. These factors were used to develop a risk score for CKD in HIV infection, externally validated, that has direct clinical relevance for patients and clinicians to weigh the benefits of certain antiretrovirals against the risk of CKD and to identify those at greatest risk of CKD.Peer reviewe

Identification and functional characterization of novel phosphorylation sites in MDC1

It is crucial for every cell of an organism to sense and repair defects that occur in its genetic material. Cells are constantly exposed to DNA-damaging agents from exogenous and endogenous sources such as the Sun’s radiation or metabolically produced free oxygen radicals. The mechanisms to deal with DNA damage consist of repair systems as well as kinase-dependent signaling pathways (so-called DNA damage checkpoints) that delay or arrest cell cycle progression. The response to DNA damage also includes alteration of transcription and regulation of apoptosis. This complex signaling network is referred to as the DNA damage response (DDR). Of all types of DNA damage, DNA double strand breaks (DSBs) pose the greatest challenge to cells. If DSBs remain un-repaired or are repaired incorrectly, the genome can suffer translocations and deletions, which may ultimately lead to genome instability and harmful diseases such as cancer. In response to DSBs, the MRE11/RAD50/NBS1 (MRN) complex acts as sensor and activates the proximal kinases ATM and DNA-PKcs. These kinases phosphorylate various target proteins including the histone variant H2AX. MDC1 is an essential mediator/adaptor protein that directly binds to phosphorylated H2AX ( H2AX) through its tandem BRCT domains. In addition, MDC1 is necessary for the amplification of the signal and for establishing a chromatin environment that is proficient for efficient repair of DSBs. In this study, we describe the molecular mechanism of MDC1-mediated accumulation and retention of the MRN complex in chromatin regions flanking DSBs. MDC1 is constitutively phosphorylated at multiple conserved SDT motifs by Casein kinase 2 (CK2). The MRN accumulation is achieved through direct binding of the FHA and BRCT tandem domain of NBS1 to the phosphorylated SDT motifs of MDC1. Mutations in conserved amino acids within the NBS1 FHA and BRCT domains, which are essential for the binding to the phospho-epitope, revealed that the accumulation of MRN in IRIF is not required for the activation of the G2/M checkpoint response. Interestingly, while cells containing a mutation in the BRCT domain of NBS1 had only a minor G2/M checkpoint defect, cells harboring a NBS1 mutated in its FHA domain showed a clear G2/M checkpoint defect at low doses of irradiation. These results suggest an additional function of the FHA domain of NBS1 in checkpoint signaling. During the search for ATM-dependent phosphorylation sites in MDC1 we identified the Thr4 residue to be targeted in a DNA damage specific manner. Interestingly, this residue bound to the FHA domain of MDC1 itself leading to dimerization of the protein. This interaction was confirmed by isothermal titration calorimetry (ITC) and X-ray crystallography, revealing that the FHA domains of two MDC1 molecules associate in a head-to-tail orientation in a DNA damage-dependent manner. These findings add MDC1 to the growing list of mediator/adaptor proteins that show dimerization and/or oligomerization. In summary, this work thus contributes substantially to our understanding of the mechanisms by which the mediator/adaptor protein MDC1 functions in the mammalian DDR. Es ist von entscheidender Bedeutung für jede Zelle eines Organismus, Defekte im Genom zu erkennen und zu reparieren. Zellen sind andauernd Substanzen ausgesetzt, welche die DNS beschädigen. Diese können von der Umwelt (z.B. Sonnenstrahlung) oder durch den Organismus selbst (z.B. freie Sauerstoffradikale) produziert werden. Die Mechanismen, um auf solche DNS Schäden zu reagieren, bestehen aus Reparatur-systemen sowie Kinase abhängigen Signalwegen (sogenannte Kontrollpunkte), welche den Zellzyklus verzögern oder anhalten. Die zelluläre Antwort auf Schäden in der DNS beinhaltet ausserdem die Regulierung der Transkription und des programmierten Zelltodes (Apoptose). Von allen Arten von Schäden der DNS stellt der Doppelstrangbruch die grösste Gefahr für eine Zelle dar. Wenn Doppelstrangbrüche nicht erkannt oder falsch repariert werden, kann das Genom durch Translokationen und Deletionen verändert werden. Diese Veränderungen können zu Instabilität des Genoms und zu gefährlichen Krankheiten (z.B. Krebs) führen. Der MRE11/RAD50/NBS1 (MRN) Komplex dient als Sensor für die Erkennung von Doppelstrangbrüchen. Er aktiviert die Kinasen ATM und DNA-PKcs, welche verschiedene Proteine phosphorylieren, darunter auch die Histon Variante H2AX. MDC1 ist ein essenzielles Adapterprotein, welches via seine BRCT Domänen direkt an phosphoryliertes H2AX bindet. Im Weiteren ermöglicht MDC1 die Rekrutierung von verschiedenen Proteinen, welche für die Reparatur und die Signalübertragung wichtig sind. Dadurch hilft MDC1 bei der Bildung einer Chromatinumgebung, welche die effiziente Reparatur von Doppelstrangbrüchen ermöglicht. In dieser Arbeit beschreiben wir den molekularen Mechanismus, wie MDC1 den MRN Komplex bindet und zu Doppelstrangbrüchen rekrutiert. MDC1 wird in mehreren SDT Motifen durch die Casein Kinase 2 konstitutiv phosphoryliert. Die Akkumulierung von MRN erfolgt durch die direkte Bindung der FHA und BRCT Domänen von NBS1 an die phosphorylierten SDT Motife von MDC1. Mutationen in konservierten Aminosäuren innerhalb der FHA und BRCT Domänen von NBS1 zeigten, dass die Bindung von MRN nicht für die Aktivierung des G2/M Kontrollpunktes verantwortlich ist. Interessanterweise hatten nur die Zellen mit einer Mutation in der FHA Domäne von NBS1 einen Defekt im G2/M Kontrollpunkt, nicht aber die Zellen mit einer Mutation in der BRCT Domäne von NBS1. Diese Resultate deuten auf eine zusätzliche Funktion der FHA Domäne von NBS1 im Signalweg nach DNS Schäden hin. Die Suche nach ATM abhängigen Phosphorylierungs-Stellen in MDC1 zeigte, dass die Aminosäure Thr4 als Folge von DNS-Schäden phosphoryliert wird. Diese Aminosäure bindet an die FHA Domäne von MDC1, was zu einer Dimerisierung des Proteins führt. Diese Bindung konnte mit Hilfe von Isothermaler Titrations Kalorimetrie sowie Röntgenstrahl-Kristallographie bestätigt werden. Dabei stellten wir fest, dass die FHA Domänen von zwei MDC1 Molekülen in umgekehrter Orientierung aneinander binden. Diese Resultate fügen MDC1 zur wachsenden Liste von Adapterproteinen hinzu, welche Dimere oder Oligomere bilden können. Insgesamt trägt diese Arbeit substanziell zu unserem Verständnis der Mechanismen bei, wie das Adapterprotein MDC1 in der zellulären Antwort auf Schäden in der DNS wirkt

A divalent FHA/BRCT-binding mechanism couples the MRE11-RAD50-NBS1 complex to damaged chromatin

The MRE11-RAD50-NBS1 (MRN) complex accumulates at sites of DNA double-strand breaks in large chromatin domains flanking the lesion site. The mechanism of MRN accumulation involves direct binding of the Nijmegen breakage syndrome 1 (NBS1) subunit to phosphorylated mediator of the DNA damage checkpoint 1 (MDC1), a large nuclear adaptor protein that interacts directly with phosphorylated H2AX. NBS1 contains an FHA domain and two BRCT domains at its amino terminus. Here, we show that both of these domains participate in the interaction with phosphorylated MDC1. Point mutations in key amino acid residues of either the FHA or the BRCT domains compromise the interaction with MDC1 and lead to defects in MRN accumulation at sites of DNA damage. Surprisingly, only mutation in the FHA domain, but not in the BRCT domains, yields a G2/M checkpoint defect, indicating that MDC1-dependent chromatin accumulation of the MRN complex at sites of DNA breaks is not required for G2/M checkpoint activation

Tritrichomonas foetus: Prevalence study in naturally mating bulls in Switzerland

Switzerland is officially free from bovine Tritrichomonas foetus. While bulls used for artificial insemination (AI) are routinely examined for this pathogen, bulls engaged in natural mating, as well as aborted fetuses, are only very sporadically investigated, indicating that the disease awareness for bovine tritrichomoniasis is low. Natural mating in cattle is becoming increasingly popular in Switzerland. Accordingly, a re-introduction/re-occurrence of T. foetus in cattle seems possible either via resurgence from a yet unknown bovine reservoir, or via importation of infected cattle. The low disease awareness for bovine tritrichomoniasis might favor an unnoticed re-establishment of T. foetus in the Swiss cattle population. The aim of our study was thus to search for the parasite, and if found, to assess the prevalence of bovine T. foetus in Switzerland. We included (1) bulls over two years of age used in natural mating and sent to slaughter, (2) bulls used for natural service in herds with or without fertility problems and (3) aborted fetuses. Furthermore, the routinely examined bulls used for AI (4) were included in this study. In total, 1362 preputial samples from bulls and 60 abomasal fluid samples of aborted fetuses were analyzed for the presence of T. foetus by both in vitro cultivation and molecular analyses. The parasite could not be detected in any of the samples, indicating that the maximal prevalence possibly missed was about 0.3% (95% confidence). Interestingly, in preputial samples of three bulls of category 1, apathogenic Tetratrichomonas sp. was identified, documenting a proof-of-principle for the methodology used in this study