Structural Insights into the Mechanism of Base Excision by MBD4

DNA glycosylases remove damaged or modified nucleobases by cleaving the N-glycosyl bond and the correct nucleotide is restored through subsequent base excision repair. In addition to excising threatening lesions, DNA glycosylases contribute to epigenetic regulation by mediating DNA demethylation and perform other important functions. However, the catalytic mechanism remains poorly defined for many glycosylases, including MBD4 (methyl-CpG binding domain IV), a member of the helix-hairpin-helix (HhH) superfamily. MBD4 excises thymine from G·T mispairs, suppressing mutations caused by deamination of 5-methylcytosine, and it removes uracil and modified uracils (e.g., 5-hydroxymethyluracil) mispaired with guanine. To investigate the mechanism of MBD4 we solved high-resolution structures of enzyme-DNA complexes at three stages of catalysis. Using a non-cleavable substrate analog, 2′-deoxy-pseudouridine, we determined the first structure of an enzyme-substrate complex for wild-type MBD4, which confirms interactions that mediate lesion recognition and suggests that a catalytic Asp, highly conserved in HhH enzymes, binds the putative nucleophilic water molecule and stabilizes the transition state. Observation that mutating the Asp (to Gly) reduces activity by 2700-fold indicates an important role in catalysis, but probably not one as the nucleophile in a double-displacement reaction, as previously suggested. Consistent with direct-displacement hydrolysis, a structure of the enzyme-product complex indicates a reaction leading to inversion of configuration. A structure with DNA containing 1-azadeoxyribose models a potential oxacarbenium-ion intermediate and suggests the Asp could facilitate migration of the electrophile towards the nucleophilic water. Finally, the structures provide detailed snapshots of the HhH motif, informing how these ubiquitous metal-binding elements mediate DNA binding.     

Dynamic and Reversible Aggregation of the Human CAP Superfamily Member GAPR-1 in Protein Inclusions in Saccharomyces cerevisiae

Many proteins that can assemble into higher order structures termed amyloids can also concentrate into cytoplasmic inclusions via liquid–liquid phase separation. Here, we study the assembly of human Golgi-Associated plant Pathogenesis Related protein 1 (GAPR-1), an amyloidogenic protein of the Cysteine-rich secretory proteins, Antigen 5, and Pathogenesis-related 1 proteins (CAP) protein superfamily, into cytosolic inclusions in Saccharomyces cerevisiae. Overexpression of GAPR-1-GFP results in the formation GAPR-1 oligomers and fluorescent inclusions in yeast cytosol. These cytosolic inclusions are dynamic and reversible organelles that gradually increase during time of overexpression and decrease after promoter shut-off. Inclusion formation is, however, a regulated process that is influenced by factors other than protein expression levels. We identified N-myristoylation of GAPR-1 as an important determinant at early stages of inclusion formation. In addition, mutations in the conserved metal-binding site (His54 and His103) enhanced inclusion formation, suggesting that these residues prevent uncontrolled protein sequestration. In agreement with this, we find that addition of Zn²⁺ metal ions enhances inclusion formation. Furthermore, Zn²⁺ reduces GAPR-1 protein degradation, which indicates stabilization of GAPR-1 in inclusions. We propose that the properties underlying both the amyloidogenic properties and the reversible sequestration of GAPR-1 into inclusions play a role in the biological function of GAPR-1 and other CAP family members.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Structural Insight into Chromatin Recognition by Multiple Domains of the Tumor Suppressor RBBP1

Retinoblastoma-binding protein 1 (RBBP1) is involved in gene regulation, epigenetic regulation, and disease processes. RBBP1 contains five domains with DNA-binding or histone-binding activities, but how RBBP1 specifically recognizes chromatin is still unknown. An AT-rich interaction domain (ARID) in RBBP1 was proposed to be the key region for DNA-binding and gene suppression. Here, we first determined the solution structure of a tandem PWWP-ARID domain mutant of RBBP1 after deletion of a long flexible acidic loop L12 in the ARID domain. NMR titration results indicated that the ARID domain interacts with DNA with no GC- or AT-rich preference. Surprisingly, we found that the loop L12 binds to the DNA-binding region of the ARID domain as a DNA mimic and inhibits DNA binding. The loop L12 can also bind weakly to the Tudor and chromobarrel domains of RBBP1, but binds more strongly to the DNA-binding region of the histone H2A-H2B heterodimer. Furthermore, both the loop L12 and DNA can enhance the binding of the chromobarrel domain to H3K4me3 and H4K20me3. Based on these results, we propose a model of chromatin recognition by RBBP1, which highlights the unexpected multiple key roles of the disordered acidic loop L12 in the specific binding of RBBP1 to chromatin.     

A Comparison of Bone Mineral Density and Its Predictors in HIV-Infected and HIV-Uninfected Older Men

ObjectiveBone health in older individuals with HIV infection has not been well studied. This study aimed to compare bone mineral density (BMD), trabecular bone score (TBS), and bone markers between HIV-infected men and age- and body mass index (BMI)-matched HIV-uninfected men aged ≥60 years. We investigated the associations of risk factors related to fracture with BMD, TBS, and bone markers in HIV-infected men.MethodsThis cross-sectional study included 45 HIV-infected men receiving antiretroviral therapy and 42 HIV-uninfected men. Medical history, BMD and TBS measurements, and laboratory tests related to bone health were assessed in all the participants. HIV-related factors known to be associated with bone loss were assessed in the HIV-infected men.ResultsThe mean BMD, TBS, and osteopenia or osteoporosis prevalence were similar among the cases and controls. The HIV-infected men had significantly higher mean N-terminal propeptide of type 1 procollagen and C-terminal cross-linking telopeptide of type I collagen levels. Stepwise multiple linear regression analysis demonstrated that low BMI (lumbar spine, P = .015; femoral neck, P = .018; and total hip, P = .005), high C-terminal cross-linking telopeptide of type I collagen concentration (total hip, P = .042; and TBS, P = .010), and low vitamin D supplementation (TBS, P = .035) were independently associated with low BMD and TBS.ConclusionIn older HIV-infected men with a low fracture risk, the mean BMD and TBS were similar to those of the age- and BMI-matched controls. The mean bone marker levels were higher in the HIV group. Traditional risk factors for fracture, including low BMI, high C-terminal cross-linking telopeptide of type I collagen level, and low vitamin D supplementation, were significant predictors of low BMD and TBS.     

Plasmid profiles and antibiotic susceptibility of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7

Abstract This paper describes the plasmid profiles obtained for 73 of 96 field isolates of Haemophilus pleuropneumoniae serotypes 1, 3, 5, and 7. We also characterized the antibiotic susceptibilities of these 96 isolates. Because of the high proportion of isolates resistant to some of the antibiotics, no conclusions can be drawn as to the role of plasmids in antibiotic resistance.     

Evidence for inhibitory SH groups in the thiol activated K:Cl cotransporter of low K sheep red blood cells

The monofunctional thiol reagents N-ethylmaleimide (NEM) and methyl methanethiosulfonate (MMTS) stimulate ouabain resistant (OR) electroneutral K:Cl cotransport in LK sheep red blood cells at low, but not at high concentrations. Diamide (DM), on the other hand, only stimulates OR K:Cl flux (Lauf, P.K., J. Memb. Biol. 101: 179–188, 1988). The DM stimulated K:Cl cotransport was decreased toward the control value prior to DM stimulation when NEM or MMTS were added, subsequently. The inhibitory effect was dependent on the compound's concentration and exposure time and, in the case of MMTS, was reversed upon addition of dithiothreitol (DTT). The inhibition was more prominent when NEM treatment was performed at pH 8.0 and disappeared at pH 6.0. In contrast the NEM stimulatory effect was most effective when the pH of NEM treatment was 6.0 (Bauer, J. & Lauf, P.K., J. Memb. Biol. 73: 257–261, 1983). The results suggest the existence of additional, however, inhibitory thiol groups in the already thiol-activated K:Cl cotransporter, with a different pKa value and a lower affinity for NEM or MMTS as compared to the stimulatory thiol groups. Like the activating thiols, the inhibitory sulfhydryls appeared to be inaccessible to non-penetrating thiol reagents and hence, must be located deeper within the red cell membrane.     

An estimate of the physical distance between two linked markers inHaemophilus influenzae

Using DNA clones, the physical distance between the linked genesnov andstr inHaemophilus influenzae was estimated. Although none of the cloned inserts contained both the markers, pJ1-8Str^R 13 (insert of 18·7 kb) includedstr gene at one end and part ofnov gene at the other end of the insert. By EcoRI restriction analysis and by Southern hybridization, the distance between the two EcoRI sites, cutting at which inactivates the two genes, was estimated to be 17·7 kb. A single continuous EcoRI fragment (containing 4EcoRI sites within it) carrying both the genes intact would need to be 20·4 kb in size. These estimates were confirmed independently using different clones ofnov ^r andstr ^r alleles as probes for hybridization with BamHI-digested chromosomal DNA.     

Toxic effects of 2-methyl-4-dimethylaminoazobenzene in normal and partially hepatectomized rats

In order to obtain a more precise definition of the conditions under which 2-methyl-4-dimethylaminoazobenzene (2-Me-DAB) and liver cell proliferation play a role in the initiation of hepatocarcinogenesis, the toxicity of 2-Me-DAB for normal and partially hepatectomized rats was investigated. Continuous feeding of a basal low protein, low riboflavin diet supplemented with 2-Me-DAB was found to be highly toxic for male albino rats. All animals fed on such a diet died before 200 days. Sham operation and partial hepatectomy (PH) at 30 days of 2-Me-DAB feeding reduced the median survival time from 122 days to 107 and 94 days, respectively. Transfer to the basal diet after 30 days of 2-Me-DAB feeding and PH prolonged the median survival time to 216 days while 97% of the rats returned to the normal complete diet after the same treatments survived for more than 300 days. 2-Me-DAB was not necrogenic and there was no evidence of reparative proliferation or hepatic tumor formation in any group. Feeding rats with the 2-Me-DAB containing diet for 1 month delayed and strongly inhibited the mitotic response of the liver to the stimulus of partial hepatectomy. This is the result of a blockage of the cells in G1 as revealed by the fact that only 1% of the hepatocytes became labeled when 2-Me-DAB fed animals were injected with tritiated thymidine prior to sacrifice at 24 h post-hepatectomy, as compared to 40% in rats fed the normal or the control basal diet. This inhibitory effect of 2-Me-DAB is reversible however since rats returned to the normal diet for 1 or 2 months after 2-Me-DAB feeding showed percentages of mitoses and labeling indices comparable to those of control animals following PH. The number of abnormal mitoses was high (13%) in regenerating livers of rats fed 2-Me-DAB and the lesions responsible for this effect are apparently not repaired since 2-Me-DAB fed rats partially hepatectomized after being transferred to the normal diet for 1 or 2 months showed the same number of mitotic irregularities. The present results suggest that assays with 2-Me-DAB as 'pure initiator' or agent of selective toxicity should be pursued in attempts to improve existing experimental models of hepatocarcinogenesis.     

Detection of microbial surface antigens that bind Lewisa antigen

Abstract There is evidence that the Lewis^a blood group antigen is one of the receptors for a number of potentially pathogenic microorganisms. To determine how widely distributed the microbial adhesins are that bind this antigen, anti-idiotypic antibodies produced against monoclonal anti-Lewis^a were used in coagglutination assays to screen a variety of species. The following were agglutinated: 7/7 strains of Staphylococcus aureus ; 10/19 (53%) strains of Neisseria meningitidis ; 8/13 (62%) strains of Haemophilus influenzae ; 1/3 strains of Helicobacter pylori ; 1/2 strains of Neisseria gonorrhoeae ; 1/2 strains of Candida albicans . The application of the anti-idiotypic antibodies to studies of host cell receptors, isolation of adhesins and development of new epidemiological typing reagents is discussed.