22 research outputs found
Hybrids of the bHLH and bZIP Protein Motifs Display Different DNA-Binding Activities In Vivo vs. In Vitro
Minimalist hybrids comprising the DNA-binding domain of bHLH/PAS (basic-helix-loop-helix/Per-Arnt-Sim) protein Arnt fused to the leucine zipper (LZ) dimerization domain from bZIP (basic region-leucine zipper) protein C/EBP were designed to bind the E-box DNA site, CACGTG, targeted by bHLHZ (basic-helix-loop-helix-zipper) proteins Myc and Max, as well as the Arnt homodimer. The bHLHZ-like structure of ArntbHLH-C/EBP comprises the Arnt bHLH domain fused to the C/EBP LZ: i.e. swap of the 330 aa PAS domain for the 29 aa LZ. In the yeast one-hybrid assay (Y1H), transcriptional activation from the E-box was strong by ArntbHLH-C/EBP, and undetectable for the truncated ArntbHLH (PAS removed), as detected via readout from the HIS3 and lacZ reporters. In contrast, fluorescence anisotropy titrations showed affinities for the E-box with ArntbHLH-C/EBP and ArntbHLH comparable to other transcription factors (Kd 148.9 nM and 40.2 nM, respectively), but only under select conditions that maintained folded protein. Although in vivo yeast results and in vitro spectroscopic studies for ArntbHLH-C/EBP targeting the E-box correlate well, the same does not hold for ArntbHLH. As circular dichroism confirms that ArntbHLH-C/EBP is a much more strongly α-helical structure than ArntbHLH, we conclude that the nonfunctional ArntbHLH in the Y1H must be due to misfolding, leading to the false negative that this protein is incapable of targeting the E-box. Many experiments, including protein design and selections from large libraries, depend on protein domains remaining well-behaved in the nonnative experimental environment, especially small motifs like the bHLH (60â70 aa). Interestingly, a short helical LZ can serve as a folding- and/or solubility-enhancing tag, an important device given the focus of current research on exploration of vast networks of biomolecular interactions
The N-end rule pathway is mediated by a complex of the RING-type Ubr1 and HECT-type Ufd4 ubiquitin ligases
Processos de democracia direta: sim ou nĂŁo? Os argumentos clĂĄssicos Ă luz da teoria e da prĂĄtica
Regularmente surgem controvĂ©rsias sobre os processos de democracia direta, dos quais os mecanismos mais frequentes sĂŁo a iniciativa popular, o plebiscito e o referendo. Por um lado, hĂĄ autores que defendem a posição de que essas instituiçÔes tornam o jogo polĂtico mais lento, caro, confuso e ilegĂtimo; outros defendem a posição contrĂĄria e argumentam que processos de democracia direta sĂŁo fundamentais para os cidadĂŁos e a qualidade da democracia. O presente estudo analisa esse tema em torno de sete questĂ”es, baseadas em consideraçÔes teĂłricas e pesquisas empĂricas: 1. A questĂŁo entre o minimalismo e o maximalismo democrĂĄtico; 2. A concorrĂȘncia entre maioria e minoria; 3. A concorrĂȘncia entre as instituiçÔes representativas e os processos de democracia direta; 4. A questĂŁo da competĂȘncia dos cidadĂŁos; 5. A questĂŁo dos efeitos colaterais dos processos de democracia direta; 6. A questĂŁo do tamanho do eleitorado; 7. A questĂŁo dos custos dos processos de democracia direta. As sete questĂ”es sĂŁo analisadas a partir de uma revisĂŁo bibliogrĂĄfica que considera tanto fontes nacionais como internacionais. O estudo mostra que os processos de democracia direta podem ser um complemento para as instituiçÔes representativas em um sistema democrĂĄtico. O bom desempenho dos plebiscitos, referendos e iniciativas populares depende tanto da regulamentação destes como tambĂ©m do desempenho das outras instituiçÔes polĂticas e da situação socioeconĂŽmica de um paĂs. O estudo permite ampliar e aprofundar o debate sobre processos de democracia direta no Brasil
Auswirkung von Quoren und Themenrestriktionen bei kommunalen BĂŒrgerbegehren im LĂ€ndervergleich
Post-translational selective intracellular silencing of acetylated proteins with de novo selected intrabodies
The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells