Quantitative analysis of the binding affinity of poly(ADP-ribose) to specific binding proteins as a function of chain length

Poly(ADP-ribose) (PAR) is synthesized by poly(ADP-ribose) polymerases in response to genotoxic stress and interacts non-covalently with DNA damage checkpoint and repair proteins. Here, we present a variety of techniques to analyze this interaction in terms of selectivity and affinity. In vitro synthesized PAR was end-labeled using a carbonyl-reactive biotin analog. Binding of HPLC-fractionated PAR chains to the tumor suppressor protein p53 and to the nucleotide excision repair protein XPA was assessed using a novel electrophoretic mobility shift assay (EMSA). Long ADP-ribose chains (55-mer) promoted the formation of three specific complexes with p53. Short PAR chains (16-mer) were also able to bind p53, yet forming only one defined complex. In contrast, XPA did not interact with short polymer, but produced a single complex with long PAR chains (55-mer). In addition, we performed surface plasmon resonance with immobilized PAR chains, which allowed establishing binding constants and confirmed the results obtained by EMSA. Taken together, we developed several new protocols permitting the quantitative characterization of PAR–protein binding. Furthermore, we demonstrated that the affinity of the non-covalent PAR interactions with specific binding proteins (XPA, p53) can be very high (nanomolar range) and depends both on the PAR chain length and on the binding protein

Poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins modulates splicing

The biological functions of poly(ADP-ribosyl)ation of heterogeneous nuclear ribonucleoproteins (hnRNPs) are not well understood. However, it is known that hnRNPs are involved in the regulation of alternative splicing for many genes, including the Ddc gene in Drosophila. Therefore, we first confirmed that poly(ADP-ribose) (pADPr) interacts with two Drosophila hnRNPs, Squid/hrp40 and Hrb98DE/hrp38, and that this function is regulated by Poly(ADP-ribose) Polymerase 1 (PARP1) and Poly(ADP-ribose) Glycohydrolase (PARG) in vivo. These findings then provided a basis for analyzing the role of pADPr binding to these two hnRNPs in terms of alternative splicing regulation. Our results showed that Parg null mutation does cause poly(ADP-ribosyl)ation of Squid and hrp38 protein, as well as their dissociation from active chromatin. Our data also indicated that pADPr binding to hnRNPs inhibits the RNA-binding ability of hnRNPs. Following that, we demonstrated that poly(ADP-ribosyl)ation of Squid and hrp38 proteins inhibits splicing of the intron in the Hsrω-RC transcript, but enhances splicing of the intron in the Ddc pre-mRNA. Taken together, these findings suggest that poly(ADP-ribosyl)ation regulates the interaction between hnRNPs and RNA and thus modulates the splicing pathways

ADP-ribose polymers localized on Ctcf–Parp1–Dnmt1 complex prevent methylation of Ctcf target sites

PARylation [poly(ADP-ribosyl)ation] is involved in the maintenance of genomic methylation patterns through its control of Dnmt1 [DNA (cytosine-5)-methyltransferase 1] activity. Our previous findings indicated that Ctcf (CCCTC-binding factor) may be an important player in key events whereby PARylation controls the unmethylated status of some CpG-rich regions. Ctcf is able to activate Parp1 [poly(ADP-ribose) polymerase 1], which ADP-ribosylates itself and, in turn, inhibits DNA methylation via non-covalent interaction between its ADP-ribose polymers and Dnmt1. By such a mechanism, Ctcf may preserve the epigenetic pattern at promoters of important housekeeping genes. The results of the present study showed Dnmt1 as a new protein partner of Ctcf. Moreover, we show that Ctcf forms a complex with Dnmt1 and PARylated Parp1 at specific Ctcf target sequences and that PARylation is responsible for the maintenance of the unmethylated status of some Ctcf-bound CpGs. We suggest a mechanism by which Parp1, tethered and activated at specific DNA target sites by Ctcf, preserves their methylation-free status

Proteome-wide identification of poly(ADP-ribose) binding proteins and poly(ADP-ribose)-associated protein complexes

Poly(ADP-ribose) (pADPr) is a polymer assembled from the enzymatic polymerization of the ADP-ribosyl moiety of NAD by poly(ADP-ribose) polymerases (PARPs). The dynamic turnover of pADPr within the cell is essential for a number of cellular processes including progression through the cell cycle, DNA repair and the maintenance of genomic integrity, and apoptosis. In spite of the considerable advances in the knowledge of the physiological conditions modulated by poly(ADP-ribosyl)ation reactions, and notwithstanding the fact that pADPr can play a role of mediator in a wide spectrum of biological processes, few pADPr binding proteins have been identified so far. In this study, refined in silico prediction of pADPr binding proteins and large-scale mass spectrometry-based proteome analysis of pADPr binding proteins were used to establish a comprehensive repertoire of pADPr-associated proteins. Visualization and modeling of these pADPr-associated proteins in networks not only reflect the widespread involvement of poly(ADP-ribosyl)ation in several pathways but also identify protein targets that could shed new light on the regulatory functions of pADPr in normal physiological conditions as well as after exposure to genotoxic stimuli

A methodology for modeling the cost and duration of concrete highway bridges

Accurately estimating the cost and duration of bridge projects is essential for optimally budgeting money and time for construction. An investigation of the current procedure used by the Transportation Department of the State of Indiana showed that it does not satisfy these important requirements. This research concentrated on analyzing an alternate methodology for optimizing cost estimating. In addition, the work offers a better strategy for estimating the duration of project construction. Three types of concrete highway bridge projects were analyzed. The projects were divided into five categories of work packages. These categories are Substructure, Superstructure, Road Approach, Traffic, and Other. The cost of each category was modeled by applying multiple regression statistical techniques. The regression analysis considered two types of models: the geometric, which considers the physical aspects of the projects; and the material quantities, which consider the major material items that integrate the models. The material quantities model was more precise for the Substructure category, while the geometric model was more appropriate for the Superstructure and the category Other. A combination of the geometric and the material quantities model was used to calculate the cost in the Approach category. The category Traffic was found to be dependent neither on the dimensions nor on the material quantities of the bridge projects. Duration models were analyzed with the same analytical approach. In this case, the geometric model was used for modeling the duration of the Superstructure and the Approach, while a combination of the geometric and the material quantities models was more representative for the Substructure duration. Although the duration of the projects cannot be obtained the same way as the costs can (by simply adding the individual category durations), the sum of the durations represents the maximum completion time period required to complete a project. The construction of the project allows some activities to overlap, so the categories may similarly overlap. The rate of overlapping, if any, is up to the contractor to determine. The analysis of this overlapping showed that there could be an exponential relation between the actual project duration and the ratio of straight/actual duration

Levantamento das emissões de gases de efeito estufa na construção de um condomínio residencial em série na cidade de Curitiba

Orientador: Prof. Dr. José de Almendra Freitas JuniorMonografia (especialização) - Universidade Federal do Paraná, Setor de Ciências Agrárias, Curso de Especialização em Projetos Sustentáveis, Mudanças Climáticas e Mercado de CarbonoInclui referências: p. 62-68Resumo: Importante setor da sociedade, a construção civil é responsável pela infraestrutura das cidades. Seus impactos, porém podem ser observados na enorme quantidade de recursos naturais utilizados e na geração de resíduos sólidos municipais na ordem de 60%. Uma das consequências da magnitude no uso de recursos é a grande emissão de gases de efeito estufa pela fabricação e transporte de materiais incorporados em edificações, em que o principal responsável é o dióxido de carbono. Com isso, o presente estudo teve como objetivo o levantamento de emissões de gases de efeito estufa na construção de um condomínio residencial com 17 unidades residenciais na cidade de Curitiba. Como resultado encontrou-se para uma unidade residencial um fator de 397,87 kgCO2eq/m² e um valor total para o condomínio de 1.002.757,22 kgCO2eq, sendo 71% das emissões originadas na realização de estruturas, alvenarias e revestimentos com argamassa, enquanto emissões por decomposição da madeira tiveram participação de 4% do total. Atuando na mitigação de emissões, os elementos de madeira armazenadores de carbono, como portas e forros, e materiais com alto índice de reciclagem, como aço e alumínio, contribuíram na redução de 3,7% dos gases de efeito estufa emitidos. Recomenda-se por fim, maior eficiência no uso de recursos e, maior uso de materiais de madeira de uso definitivo, para armazenamento de carbono, e produtos com baixa emissão incorporada pelo seu processo produtivo como forma de mitigação às emissões de gases de efeito estufa