RESUMO
Ribosome biogenesis is a key process in all eukaryotic cells that requires hundreds of ribosome biogenesis factors (RBFs), which are essential to build the mature ribosomes consisting of proteins and rRNAs. The processing of the required rRNAs has been studied extensively in yeast and mammals, but in plants much is still unknown. In this study, we focused on a RBF from A. thaliana that we named NUCLEOLAR RNA CHAPERONE-LIKE 1 (NURC1). NURC1 was localized in the nucleolus of plant cell nuclei, and other plant RBF candidates shared the same localization. SEC-SAXS experiments revealed that NURC1 has an elongated and flexible structure. In addition, SEC-MALLS experiments confirmed that NURC1 was present in its monomeric form with a molecular weight of around 28 kDa. RNA binding was assessed by performing microscale thermophoresis with the Arabidopsis internal transcribed spacer 2 (ITS2) of the polycistronic pre-rRNA precursor, which contains the 5.8S, 18S, and 25S rRNA. NURC1 showed binding activity to the ITS2 with a dissociation constant of 228 nM and exhibited RNA chaperone-like activity. Our data suggested that NURC1 may have a function in pre-rRNA processing and thus ribosome biogenesis.
Assuntos
Arabidopsis , Proteínas de Plantas , Animais , Proteínas Nucleares , Espalhamento a Baixo Ângulo , Difração de Raios X , Arabidopsis/genética , RNA , Precursores de RNA , MamíferosRESUMO
The trimeric CCAAT-binding NF-Y is a "pioneer" Transcription Factor -TF- known to cooperate with neighboring TFs to regulate gene expression. Genome-wide analyses detected a precise stereo-alignment -10/12 bp- of CCAAT with E-box elements and corresponding colocalization of NF-Y with basic-Helix-Loop-Helix (bHLH) TFs. We dissected here NF-Y interactions with USF1 and MAX. USF1, but not MAX, cooperates in DNA binding with NF-Y. NF-Y and USF1 synergize to activate target promoters. Reconstruction of complexes by structural means shows independent DNA binding of MAX, whereas USF1 has extended contacts with NF-Y, involving the USR, a USF-specific amino acid sequence stretch required for trans-activation. The USR is an intrinsically disordered domain and adopts different conformations based on E-box-CCAAT distances. Deletion of the USR abolishes cooperative DNA binding with NF-Y. Our data indicate that the functionality of certain unstructured domains involves adapting to small variation in stereo-alignments of the multimeric TFs sites.
Assuntos
DNA/metabolismo , Fatores Estimuladores Upstream/metabolismo , Regulação da Expressão Gênica , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , Domínios ProteicosRESUMO
Sarcomeres, the basic contractile units of striated muscle cells, contain arrays of thin (actin) and thick (myosin) filaments that slide past each other during contraction. The Ig-like domain-containing protein myotilin provides structural integrity to Z-discs-the boundaries between adjacent sarcomeres. Myotilin binds to Z-disc components, including F-actin and α-actinin-2, but the molecular mechanism of binding and implications of these interactions on Z-disc integrity are still elusive. To illuminate them, we used a combination of small-angle X-ray scattering, cross-linking mass spectrometry, and biochemical and molecular biophysics approaches. We discovered that myotilin displays conformational ensembles in solution. We generated a structural model of the F-actin:myotilin complex that revealed how myotilin interacts with and stabilizes F-actin via its Ig-like domains and flanking regions. Mutant myotilin designed with impaired F-actin binding showed increased dynamics in cells. Structural analyses and competition assays uncovered that myotilin displaces tropomyosin from F-actin. Our findings suggest a novel role of myotilin as a co-organizer of Z-disc assembly and advance our mechanistic understanding of myotilin's structural role in Z-discs.
Assuntos
Actinas/metabolismo , Multimerização Proteica , Sarcômeros/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/genética , Animais , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Contração Muscular/genética , Músculo Esquelético/metabolismo , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Multimerização Proteica/genética , Sarcômeros/genética , Tropomiosina/química , Tropomiosina/genética , Tropomiosina/metabolismoRESUMO
Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease.
