Deciphering cellular and molecular determinants of human DPCD protein in complex with RUVBL1/RUVBL2 AAA-ATPases.

DPCD is a protein that may play a role in cilia formation and whose absence leads to primary ciliary dyskinesia (PCD), a rare disease caused by impairment of ciliated cells. Except for high-throughput studies that identified DPCD as a possible RUVBL1 (R1) and RUVBL2 (R2) partner, no in-depth cellular, biochemical, and structural investigation involving DPCD have been reported so far. R1 and R2 proteins are ubiquitous highly conserved AAA + family ATPases that assemble and mature a plethora of macromolecular complexes and are pivotal in numerous cellular processes, especially by guaranteeing a co-chaperoning function within R2TP or R2TP-like machineries.

Structural Analysis of the Plasmodial Proteins ZNHIT3 and NUFIP1 Provides Insights into the Selectivity of a Conserved Interaction.

Malaria is a widespread and lethal disease caused by the parasites that can infect human beings through Anopheles mosquitoes. For that reason, the biology of needs to be studied to develop antimalarial treatments. By determining the three-dimensional structures of macromolecules, structural biology helps to understand the function of proteins and can reveal how interactions occur between biological partners.

The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity.

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles.

Constitutive and variable 2'-O-methylation (Nm) in human ribosomal RNA.

Epitranscriptomic modifications of stable RNAs are dynamically regulated and specific profiles of 2'-O-methylation in rRNA have been associated with distinct cancer types. However, these observations pointed out the existence of at least two distinct groups: a rather large group with constitutive rRNA Nm residues exhibiting a stable level of methylation and a more restricted set of variable modifications, giving rise to the concept of 'specialized ribosomes'. These heterogeneous ribosomes can modulate their translational properties and be key regulatory players, depending on the physiological state of the cell.

Application of NMR Spectroscopy to Determine the 3D Structure of Small Non-Coding RNAs.

Many RNA architectures were discovered to be involved in a wide range of essential biological processes in all organisms from carrying genetic information to gene expression regulation. The remarkable ability of RNAs to adopt various architectures depending on their environment enables the achievement of their myriads of biological functions. Nuclear Magnetic Resonance (NMR) is a powerful technique to investigate both their structure and dynamics.

The box C/D snoRNP assembly factor Bcd1 interacts with the histone chaperone Rtt106 and controls its transcription dependent activity.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoproteins initiates co-transcriptionally and requires the action of the assembly machinery including the Hsp90/R2TP complex, the Rsa1p:Hit1p heterodimer and the Bcd1 protein. We present genetic interactions between the Rsa1p-encoding gene and genes involved in chromatin organization including RTT106 that codes for the H3-H4 histone chaperone Rtt106p controlling H3K56ac deposition. We show that Bcd1p binds Rtt106p and controls its transcription-dependent recruitment by reducing its association with RNA polymerase II, modulating H3K56ac levels at gene body.

Optimizing the First TPR Domain of the Human SPAG1 Protein Provides Insight into the HSP70 and HSP90 Binding Properties.

Tetratricopeptide repeat domains, or TPR domains, are protein domains that mediate protein:protein interaction. As they allow contacts between proteins, they are of particular interest in transient steps of the assembly process of macromolecular complexes, such as the ribosome or the dynein arms. In this study, we focused on the first TPR domain of the human SPAG1 protein.

NOPCHAP1 is a PAQosome cofactor that helps loading NOP58 on RUVBL1/2 during box C/D snoRNP biogenesis.

The PAQosome is a large complex composed of the HSP90/R2TP chaperone and a prefoldin-like module. It promotes the biogenesis of cellular machineries but it is unclear how it discriminates closely related client proteins. Among the main PAQosome clients are C/D snoRNPs and in particular their core protein NOP58.

Box C/D snoRNPs: solid-state NMR fingerprint of an early-stage 50 kDa assembly intermediate.

Many cellular functions rely on stable protein-only or protein-RNA complexes. Deciphering their assembly mechanism is a key question in cell biology. We here focus on box C/D small nucleolar ribonucleoproteins involved in ribosome biogenesis.

Binding properties of the quaternary assembly protein SPAG1.

In cells, many constituents are able to assemble resulting in large macromolecular machineries possessing very specific biological and physiological functions, e.g. ribosome, spliceosome and proteasome.

The yeast C/D box snoRNA U14 adopts a "weak" K-turn like conformation recognized by the Snu13 core protein in solution.

Non-coding RNAs associate with proteins to form ribonucleoproteins (RNPs), such as ribosome, box C/D snoRNPs, H/ACA snoRNPs, ribonuclease P, telomerase and spliceosome to ensure cell viability. The assembly of these RNA-protein complexes relies on the ability of the RNA to adopt the correct bound conformation. K-turn motifs represent ubiquitous binding platform for proteins found in several cellular environment.

CRD Generated by pCARGHO: A New Efficient Lectin-Based Affinity Tag Method for Safe, Simple, and Low-Cost Protein Purification.

Purification of recombinant proteins remains a bottleneck for downstream processing. The authors engineered a new galectin 3 truncated form (CRD ), functionally and structurally characterized, with preserved solubility and lectinic activity. Taking advantage of these properties, the authors designed an expression vector (pCARGHO), suitable for CRD -tagged protein expression in prokaryotes.

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90.

The RPAP3-Cterminal domain identifies R2TP-like quaternary chaperones.

R2TP is an HSP90 co-chaperone that assembles important macro-molecular machineries. It is composed of an RPAP3-PIH1D1 heterodimer, which binds the two essential AAA+ATPases RUVBL1/RUVBL2. Here, we resolve the structure of the conserved C-terminal domain of RPAP3, and we show that it directly binds RUVBL1/RUVBL2 hexamers.

NMR assignment and solution structure of the external DII domain of the yeast Rvb2 protein.

We report the nearly complete H, N and C resonance assignment and the solution structure of the external DII domain of the yeast Rvb2 protein, a member of the AAA+ATPase superfamily.

Structural Features of the Box C/D snoRNP Pre-assembly Process Are Conserved through Species.

Box C/D small nucleolar ribonucleoparticles (snoRNPs) support 2'-O-methylation of several target RNAs. They share a common set of four core proteins (SNU13, NOP58, NOP56, and FBL) that are assembled on different guide small nucleolar RNAs. Assembly of these entities involves additional protein factors that are absent in the mature active particle.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis.

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins.

Structure/Function Analysis of Protein-Protein Interactions Developed by the Yeast Pih1 Platform Protein and Its Partners in Box C/D snoRNP Assembly.

In eukaryotes, nucleotide post-transcriptional modifications in RNAs play an essential role in cell proliferation by contributing to pre-ribosomal RNA processing, ribosome assembly and activity. Box C/D small nucleolar ribonucleoparticles catalyze site-specific 2'-O-methylation of riboses, one of the most prevalent RNA modifications. They contain one guide RNA and four core proteins and their in vivo assembly requires numerous factors including (HUMAN/Yeast) BCD1/Bcd1p, NUFIP1/Rsa1p, ZNHIT3/Hit1p, the R2TP complex composed of protein PIH1D1/Pih1p and RPAP3/Tah1p that bridges the R2TP complex to the HSP90/Hsp82 chaperone and two AAA+ ATPases.

Accurate protein-peptide titration experiments by nuclear magnetic resonance using low-volume samples.

NMR spectroscopy allows measurements of very accurate values of equilibrium dissociation constants using chemical shift perturbation methods, provided that the concentrations of the binding partners are known with high precision and accuracy. The accuracy and precision of these experiments are improved if performed using individual capillary tubes, a method enabling full automation of the measurement. We provide here a protocol to set up and perform these experiments as well as a robust method to measure peptide concentrations using tryptophan as an internal standard.

Protein Hit1, a novel box C/D snoRNP assembly factor, controls cellular concentration of the scaffolding protein Rsa1 by direct interaction.

Biogenesis of eukaryotic box C/D small nucleolar ribonucleoprotein particles (C/D snoRNPs) involves conserved trans-acting factors, which are proposed to facilitate the assembly of the core proteins Snu13p/15.5K, Nop58p/NOP58, Nop56p/NOP56 and Nop1p/Fibrillarin on box C/D small nucleolar RNAs (C/D snoRNAs). In yeast, protein Rsa1 acts as a platform, interacting with both the RNA-binding core protein Snu13 and protein Pih1 of the Hsp82-R2TP chaperone complex.

(1)H, (15)N and (13)C resonance assignments of the two TPR domains from the human RPAP3 protein.

We report the nearly complete (1)H, (15)N and (13)C resonance assignments of the two tetratricopeptide-repeat domains of the human RPAP3 protein, a co-chaperone of the heat-shock protein family.

(1)H, (15)N and (13)C resonance assignments of the yeast Pih1 and Tah1 C-terminal domains complex.

We report the nearly complete (1)H, (15)N and (13)C resonance assignment of the complex formed by the C-terminal domains of Pih1 and Tah1 from S. cerevisiae and evidence the folding ability of Tah1 under complex formation.

Heteronuclear NMR provides an accurate assessment of therapeutic insulin's quality.

New generations of drugs are using more and more often therapeutic proteins as the active ingredient, prompting the regulation agencies to adapt their analytical methods. Fast and unambiguous information on the secondary, tertiary and quaternary structure of the protein should be provided to assess the quality of these biodrugs. Recent developments of heteronuclear NMR methods, enabling their use on pharmaceutical formulated unlabeled proteins, provide an efficient way to perform such analysis, a feature that is illustrated here using various commercial formulations of insulins.

Unraveling complex small-molecule binding mechanisms by using simple NMR spectroscopy.

Heteronuclear NMR spectroscopy provides a unique way to obtain site-specific information about protein-ligand interactions. Usually, such studies rely on the availability of isotopically labeled proteins, thereby allowing both editing of the spectra and ligand signals to be filtered out. Herein, we report that the use of the methyl SOFAST correlation experiment enables the determination of site-specific equilibrium binding constants by using unlabeled proteins.