Unveiling the Role of Sorghum RPAP3 in the Function of R2TP Complex: Insights into Protein Assembly in Plants.

The chaperone R2TP has multiple subunits that assist in the proper folding, assembly, and stabilization of various protein complexes in cells and its study can offer valuable insights into the regulation and maintenance of protein assemblies in plant systems. The 'T' component of R2TP is Tah1 in yeast, consisting of 111 residues, while its counterpart in humans is RPAP3, with 665 residues. RPAP3 acts as a co-chaperone of Hsp90 and facilitates interactions between RUVBL proteins and other complex components, enhancing the recruitment of client proteins by the R2TP complex. These facts further underscore the relevance of studying this complex in different organisms. The putative gene corresponding to the RPAP3 in Sorghum bicolor, a monocotyledon plant, was cloned, and the protein (396 residues) purified for biochemical characterization. SbRPAP3 exists as a folded monomer and has a RPAP3 domain, which is present in human RPAP3 but absent in yeast Tah1. SbRPAP3 retains its functional capabilities, including binding with RUVBLs, Hsp90, and Hsp70. By elucidating the role of RPAP3 in plant R2TP complex, we can further comprehend the molecular mechanisms underlying plant-specific protein assembly and contribute to advancements in plant biology and biotechnological applications.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. METHODS: We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. RESULTS: ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. CONCLUSION: These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.

Escherichia coli vacuolating factor, involved in avian cellulitis, induces actin contraction and binds to cytoskeleton proteins in fibroblasts

Avian pathogenic Escherichia coli (APEC) isolated from avian cellulitis lesions produces a toxin, named Escherichia coli vacuolating factor (ECVF), that causes cell vacuolization and induces inflammatory response in broiler chicken. Methods We investigated the intracellular activities of ECVF in avian fibroblasts using fluorescence staining, electron microscopy, MTT and LDH measurements. As ECVF act specifically in avian cells, we performed blotting assay followed by mass spectrometry to better understand its initial intracellular protein recognition. Results ECVF induced actin contraction, mitochondrial damage and membrane permeability alterations. Ultrastructural analysis showed intracellular alterations, as nuclear lobulation and the presence of degraded structures inside the vacuoles. Moreover, ECVF induced cell death in fibroblasts. ECVF-biotin associates to at least two proteins only in avian cell lysates: alpha-actinin 4 and vinculin, both involved in cytoskeleton structure. Conclusion These findings demonstrated that ECVF plays an important role in avian cellulitis, markedly in initial steps of infection. Taken together, the results place this toxin as a target for drug and/or vaccine development, instead of the use of large amounts antibiotics.(AU)

Optimization of a Methodology for Quantification and Removal of Zinc Gives Insights Into the Effect of This Metal on the Stability and Function of the Zinc-Binding Co-chaperone Ydj1.

Ydj1, a class B J-protein (Hsp40) in yeast, has two zinc finger domains in each monomer and belongs to an important co-chaperone family that plays crucial roles in cells, such as recognizing and binding partially folded proteins and assisting the Hsp70 chaperone family in protein folding. Yeast cells with ydj1 deletion were less efficient at coping with zinc stress than wild-type cells, and site-directed mutagenesis studies that impair or delete the zinc finger region have confirmed the importance of this region to the function of Ydj1; however, little is known about whether the presence of zinc is critical for the function of the protein. To gain insights into the effect of zinc on the structure and function of Ydj1 without having to modify its primary structure, a method was developed and optimized to quantify and remove the zinc from the protein. Recombinant Ydj1 was produced and purified, and its zinc content was determined by ICP-MS. The result showed that two zinc atoms were bound per monomer of protein, a good indicator that all sites were saturated. To optimize the removal of the bound zinc, variations on chelating agent (EDTA, EGTA, 1,10-phenanthroline), chelator concentration, reaction time, pH, and temperature were tested. These procedures had no effect on the overall secondary structure of the protein, since no significant changes in the circular dichroism spectrum were observed. The most significant removal (91 ± 2%, n = 3) of zinc was achieved using 1,10-phenanthroline (1 × 10-3 mol L-1) at 37°C with a pH 8.5 for 24 h. Zinc removal affected the stability of the protein, as observed by a thermal-induced unfolding assay showing that the temperature at the middle of the transition (Tm) decreased from 63 ± 1°C to 60 ± 1°C after Zn extraction. In addition, the effect on the ability of Ydj1 to protect a model protein (luciferase) against aggregation was completely abolished after the Zn removal procedure. The main conclusion is that zinc plays an important role in the stability and activity of Ydj1. Additionally, the results highlight the medical importance of chaperones, as altered zinc homeostasis is implicated in many diseases, such as neurodegenerative disorders.

Proteomic characterization of the human FTSJ3 preribosomal complexes.

In eukaryotes, ribosome biogenesis involves excision of transcribed spacer sequences from the preribosomal RNA, base and ribose covalent modification at specific sites, assembly of ribosomal proteins, and transport of subunits from the nucleolus to the cytoplasm where mature ribosomes engage in mRNA translation. The biochemical reactions throughout ribosome synthesis are mediated by factors that associate transiently to the preribosomal complexes. In this work, we describe the complexes containing the human protein FTSJ3. This protein functions in association with NIP7 in ribosome synthesis and contains a putative RNA-methyl-transferase domain (FtsJ) in the N-terminal region and two uncharacterized domains in the central (DUF3381) and C-terminal (Spb1_C) regions. FLAG-tagged FTSJ3 coimmunoprecipitates both RPS and RPL proteins, ribosome synthesis factors, and proteins whose function in ribosome synthesis has not been demonstrated yet. A similar set of proteins coimmunoprecipitates with the Spb1_C domain, suggesting that FTSJ3 interaction with the preribosome complexes is mediated by the Spb1_C domain. Approximately 50% of the components of FTSJ3 complexes are shared by complexes described for RPS19, Par14, nucleolin, and NOP56. A significant number of factors are also found in complexes described for nucleophosmin, SBDS, ISG20L2, and NIP7. These findings provide information on the dynamics of preribosome complexes in human cells.