Nayef Mazloum

Direct Interaction of Proliferating Cell Nuclear Antigen with the p125 Catalytic Subunit of Mammalian DNA Polymerase δ

The formation of a complex between DNA polymerase δ (pol δ) and its sliding clamp, proliferating cell nuclear antigen (PCNA), is responsible for the maintenance of processive DNA synthesis at the leading strand of the replication fork. In this study, the ability of the p125 catalytic subunit of DNA polymerase δ to engage in protein-protein interactions with PCNA was established by biochemical and genetic methods. p125 and PCNA were shown to co-immunoprecipitate from either calf thymus or HeLa extracts, or when they were ectopically co-expressed in Cos 7 cells. Because pol δ is a multimeric protein, this interaction could be indirect. Thus, rigorous evidence was sought for a direct interaction of the p125 catalytic subunit and PCNA. To do this, the ability of recombinant p125 to interact with PCNA was established by biochemical means. p125 co-expressed with PCNA in Sf9 cells was shown to form a physical complex that can be detected on gel filtration and that can be cross-linked with the bifunctional cross-linking agent Sulfo-EGS (ethylene glycol bis (sulfosuccinimidylsuccinate)). An interaction between p125 and PCNA could also be demonstrated in the yeast two hybrid system. Overlay experiments using biotinylated PCNA showed that the free p125 subunit interacts with PCNA. The PCNA overlay blotting method was also used to demonstrate the binding of synthetic peptides corresponding to the N2 region of pol δ and provides evidence for a site on pol δ that is involved in the protein-protein interactions between PCNA and pol δ. This region contains a sequence that is a potential member of the PCNA binding motif found in other PCNA-binding proteins. These studies provide an unequivocal demonstration that the p125 subunit of pol δ interacts with PCNA.

Dss1 Interaction with Brh2 as a Regulatory Mechanism for Recombinational Repair

ABSTRACT Brh2, the BRCA2 ortholog in Ustilago maydis, enables recombinational repair of DNA by controlling Rad51 and is in turn regulated by Dss1. Interplay with Rad51 is conducted via the BRC element located in the N-terminal region of the protein and through an unrelated domain, CRE, at the C terminus. Mutation in either BRC or CRE severely reduces functional activity, but repair deficiency of the brh2 mutant can be complemented by expressing BRC and CRE on different molecules. This intermolecular complementation is dependent upon the presence of Dss1. Brh2 molecules associate through the region overlapping with the Dss1-interacting domain to form at least dimer-sized complexes, which in turn, can be dissociated by Dss1 to monomer. We propose that cooperation between BRC and CRE domains and the Dss1-provoked dissociation of Brh2 complexes are requisite features of Brh2s molecular mechanism.

Analysis of the role of the leucine zipper motif in regulating the ability of AFAP‐110 to alter actin filament integrity

AFAP‐110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self‐association stabilizing the AFAP‐110 multimers. Deletion of the Lzip motif (AFAP‐110Δlzip) reduces the stability of the AFAP‐110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP‐110 function. Substitution of the c‐Fos Lzip motif in place of the AFAP‐110 Lzip motif (AFAP‐110fos) was predicted to preserve the α‐helical structure while changing the sequence. To alter the structure of the α‐helix, a leucine to proline mutation was generated in the AFAP‐110 α‐helical Lzip motif (AFAP‐110581P), which largely preserved the sequence. The helix mutants, AFAP‐110Δlzip, AFAP‐110fos, and AFAP‐110581P, demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP‐110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto‐inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP‐110Δlzip and AFAP‐110581P were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP‐110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP‐110‐directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP‐110 to crosslink actin filaments or activate cSrc are distinct functions.

Second-End Capture in DNA Double-Strand Break Repair Promoted by Brh2 Protein of Ustilago maydis

Brh2 plays a central role in the homologous recombination system of Ustilago maydis, mediating delivery of Rad51 to single-stranded DNA. Here we report that Brh2 can pair the displaced strand of a D loop with a complementary single-stranded DNA to form a duplexed, or double, D loop. The reaction emulates the second-end capture step envisioned in models of DNA double-strand break repair. This second-end capture reaction promoted by Brh2 proceeds efficiently when performed in the presence of Rad51 under conditions that block annealing by Rad52, or when the second single-stranded DNA substrate is replaced by double-stranded DNA. In a coupled reaction that requires extension of the D loop more than 200 nt by DNA synthesis in order to reveal a complementary region, Brh2 was also able to promote second-end capture and thus model a synthesis-dependent strand-annealing mechanism.

Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair

Bacterial DNA can be damaged by reactive nitrogen and oxygen intermediates (RNI and ROI) generated by host immunity, as well as by antibiotics that trigger bacterial production of ROI. Thus a pathogen’s ability to repair its DNA may be important for persistent infection. A prominent role for nucleotide excision repair (NER) in disease caused by Mycobacterium tuberculosis (Mtb) was suggested by attenuation of uvrB-deficient Mtb in mice. However, it was unknown if Mtb’s Uvr proteins could execute NER. Here we report that recombinant UvrA, UvrB, and UvrC from Mtb collectively bound and cleaved plasmid DNA exposed to ultraviolet (UV) irradiation or peroxynitrite. We used the DNA incision assay to test the mechanism of action of compounds identified in a high-throughput screen for their ability to delay recovery of M. smegmatis from UV irradiation. 2-(5-Amino-1,3,4-thiadiazol-2-ylbenzo[f]chromen-3-one) (ATBC) but not several closely related compounds inhibited cleavage of damaged DNA by UvrA, UvrB, and UvrC without intercalating in DNA and impaired recovery of M. smegmatis from UV irradiation. ATBC did not affect bacterial growth in the absence of UV exposure, nor did it exacerbate the growth defect of UV-irradiated mycobacteria that lacked uvrB. Thus, ATBC appears to be a cell-penetrant, selective inhibitor of mycobacterial NER. Chemical inhibitors of NER may facilitate studies of the role of NER in prokaryotic pathobiology.

SIRT1 Limits Adipocyte Hyperplasia through c-Myc Inhibition

The expansion of fat mass in the obese state is due to increased adipocyte hypertrophy and hyperplasia. The molecular mechanism that drives adipocyte hyperplasia remains unknown. The NAD+-dependent protein deacetylase sirtuin 1 (SIRT1), a key regulator of mammalian metabolism, maintains proper metabolic functions in many tissues, counteracting obesity. Here we report that differentiated adipocytes are hyperplastic when SIRT1 is knocked down stably in mouse 3T3-L1 preadipocytes. This phenotype is associated with dysregulated adipocyte metabolism and enhanced inflammation. We also demonstrate that SIRT1 is a key regulator of proliferation in preadipocytes. Quantitative proteomics reveal that the c-Myc pathway is altered to drive enhanced proliferation in SIRT1-silenced 3T3-L1 cells. Moreover, c-Myc is hyperacetylated, levels of p27 are reduced, and cyclin-dependent kinase 2 (CDK2) is activated upon SIRT1 reduction. Remarkably, differentiating SIRT1-silenced preadipocytes exhibit enhanced mitotic clonal expansion accompanied by reduced levels of p27 as well as elevated levels of CCAAT/enhancer-binding protein β (C/EBPβ) and c-Myc, which is also hyperacetylated. c-Myc activation and enhanced proliferation phenotype are also found to be SIRT1-dependent in proliferating mouse embryonic fibroblasts and differentiating human SW872 preadipocytes. Reducing both SIRT1 and c-Myc expression in 3T3-L1 cells simultaneously does not induce the adipocyte hyperplasia phenotype, confirming that SIRT1 controls adipocyte hyperplasia through c-Myc regulation. A better understanding of the molecular mechanisms of adipocyte hyperplasia will open new avenues toward understanding obesity.

D-loop formation by Brh2 protein of Ustilago maydis

Brh2, the ortholog of the BRCA2 tumor suppressor in Ustilago maydis, works hand in hand with Rad51 to promote repair of DNA by homologous recombination. Previous studies established that Brh2 can stimulate DNA strand exchange by enabling Rad51 nucleoprotein filament formation on replication protein A-coated ssDNA. But, more recently, it was noted that Brh2 has an inherent DNA annealing activity, raising the notion that it might have roles in recombination in addition to or beyond the mediator function. Here, we found that Brh2 can autonomously promote the formation of D-loops in reactions with plasmid DNA and homologous single-stranded oligonucleotides. The reaction differs from that catalyzed by Rad51 in having no requirement for cofactors or preloading phase on ssDNA. D-loop formation was most effective when Brh2 was mixed with plasmid DNA before addition of single-stranded oligomer. D-loop formation catalyzed by Rad51 was also enhanced when Brh2 was premixed with plasmid DNA. Brh2 rendered defective in Rad51 interaction by mutation in the BRC element was still capable of promoting D-loop formation. However, the mutant protein was unable to enhance the Rad51-catalyzed reaction. The results suggest a model in which Brh2 binding to plasmid DNA attracts and helps capture Rad51-coated ssDNA.

Dss1 regulates interaction of Brh2 with DNA

Brh2, the BRCA2 homologue in Ustilago maydis, plays a crucial role in homologous recombination by controlling Rad51. In turn, Brh2 is governed by Dss1, an intrinsically disordered protein that forms a tight complex with the C-terminal region of Brh2. This region of the protein associating with Dss1 is highly conserved in sequence and by comparison with mammalian BRCA2 corresponds to a part of the DNA binding domain with characteristic OB folds. The N-terminal region of Brh2 harbors a less-defined but powerful DNA binding site, the activity of which is revealed upon deletion of the C-terminal region. Full-length Brh2 complexed with Dss1 binds DNA slowly, while the N-terminal fragment binds quickly. The DNA binding activity of full-length Brh2 appears to correlate with dissociation of Dss1. Addition of Dss1 to the heterotypic Brh2-Dss1 complex attenuates DNA binding activity, but not by direct competition for the N-terminal DNA binding site. Conversely, the Brh2-Dss1 complex dissociates more quickly when DNA is present. These findings suggest a model in which binding of Brh2 to DNA is subject to allosteric regulation by Dss1.

Interaction of the retinoblastoma protein (pRb) with the catalytic subunit of DNA polymerase δ (p125)

The retinoblastoma gene product (pRb) interacts with many cellular proteins to function in the control of cell division, differentiation, and apoptosis. Several pRb binding proteins complex with pRb through an amino acid sequence called the LXCXE motif. The catalytic subunit of DNA polymerase δ (p125) contains a LXCXE motif. To further study the biochemical function of this polymerase, we sought to determine if p125 interacts with pRb. Experiments using GST-pRb fusion proteins showed that p125 from breast epithelial (MCF10A) cell extracts associates with pRb. In addition, GST-p125 fusion proteins bound pRb from the same cell extracts. The pRb that associated with GST-p125 was largely unphosphorylated. Coimmunoprecipitation experiments using cell cycle synchronized cells revealed that p125 and pRb form a complex predominantly during G1 phase, the phase during which pRb is mostly unphosphorylated. In vitro phosphorylation of GST-pRb by the cyclin dependent kinases reduced the ability of p125 to associate with GST-pRb. Addition of the LXCXE containing protein SV40 large T antigen to GST-pRb blocks the ability of p125 to associate with pRb, suggesting that it may be through a LXCXE sequence by which p125 interacts with pRb. Finally, in vitro polymerase assays demonstrate that GST-pRb fusion protein stimulates DNA polymerase δ activity.

Combined metformin and insulin treatment reverses metabolically impaired omental adipogenesis and accumulation of 4-hydroxynonenal in obese diabetic patients

Objective Obesity-associated impaired fat accumulation in the visceral adipose tissue can lead to ectopic fat deposition and increased risk of insulin resistance and type 2 diabetes mellitus (T2DM). This study investigated whether impaired adipogenesis of omental (OM) adipose tissues and elevated 4-hydroxynonenal (4-HNE) accumulation contribute to this process, and if combined metformin and insulin treatment in T2DM patients could rescue this phenotype. Methods OM adipose tissues were obtained from forty clinically well characterized obese individuals during weight reduction surgery. Levels of 4-HNE protein adducts, adipocyte size and number of macrophages were determined within these tissues by immunohistochemistry. Adipogenic capacity and gene expression profiles were assessed in preadipocytes derived from these tissues in relation to insulin resistance and in response to 4-HNE, metformin or combined metformin and insulin treatment. Results Preadipocytes isolated from insulin resistant (IR) and T2DM individuals exhibited lower adipogenesis, marked by upregulation of anti-adipogenic genes, compared to preadipocytes derived from insulin sensitive (IS) individuals. Impaired adipogenesis was also associated with increased 4-HNE levels, smaller adipocytes and greater macrophage presence in the adipose tissues. Within the T2DM group, preadipocytes from combined metformin and insulin treated subset showed better in vitro adipogenesis compared to metformin alone, which was associated with less presence of macrophages and 4-HNE in the adipose tissues. Treatment of preadipocytes in vitro with 4-HNE reduced their adipogenesis and increased proliferation, even in the presence of metformin, which was partially rescued by the presence of insulin. Conclusion This study reveals involvement of 4-HNE in the impaired OM adipogenesis-associated with insulin resistance and T2DM and provides a proof of concept that this impairment can be reversed by the synergistic action of insulin and metformin. Further studies are needed to evaluate involvement of 4-HNE in metabolically impaired abdominal adipogenesis and to confirm benefits of combined metformin-insulin therapy in T2DM patients.