Introducing protein deamidation: Landmark discoveries, societal outreach, and tentative priming workflow to address deamidation.

Our current knowledge on protein deamidation results from a journey that started almost 100 years ago, when a handful of researchers first described the non-enzymatic "desamidation" of glutamine, and the effect of different anions on the catalytic rate of the reaction. Since then, the field has tremendously expended and now finds outreach in very diverse areas. In light of all the recent articles published in these areas, it seemed timely to propose an integrated review on the subject, including a short historical overview of the landmark discoveries in the field, highlighting the current global positioning of protein deamidation in biology and non-biology fields, and concluding with a workflow for those asking if a protein can deamidate, and identify the residues involved. This review is essentially intended to provide newcomers in the field with an overview of how deamidation has penetrated our society and what tools are currently at hand to identify and quantify protein deamidation.

1D continuous gel electrophoresis composition for the separation of deamidated proteins.

Deamidation is a spontaneous modification of peptides and proteins that has potent repercussions on their activity and stability in vivo and in vitro. Being able to implement easy techniques to detect and quantify protein deamidation is a major goal in this field. Here we focus on electrophoretic methods that can be deployed to assess protein deamidation. We provide an update on the use of Taurine/Glycinate as trailing ions to assist the detection of several examples of deamidated proteins, namely the small GTPases RhoA, Rac1 and Cdc42, but also the oncogene Bcl-xL and calcium-binding Calmodulin. We also report on the use of imidazole as a counter ion to improve the focusing of deamidated bands. Finally, we provide examples of how these gels proved useful to compare on full-length proteins the effect of ions and pH on the catalytic rates of spontaneous deamidation. Taken together, the electrophoretic method introduced here proves useful to screen at once the effect of various conditions of pH, ionic strength and buffer ions on protein stability. Direct applications can be foreseen to tailor buffer formulations to control the stability of proteins drug products.

Improved Electrophoretic Separation to Assist the Monitoring of Bcl-xL Post-Translational Modifications.

Bcl-xL is an oncogene of which the survival functions are finely tuned by post-translational modifications (PTM). Within the Bcl-2 family of proteins, Bcl-xL shows unique eligibility to deamidation, a time-related spontaneous reaction. Deamidation is still a largely overlooked PTM due to a lack of easy techniques to monitor Asn→Asp/IsoAsp conversions or Glu→Gln conversions. Being able to detect PTMs is essential to achieve a comprehensive description of all the regulatory mechanisms and functions a protein can carry out. Here, we report a gel composition improving the electrophoretic separation of deamidated forms of Bcl-xL generated either by mutagenesis or by alkaline treatment. Importantly, this new gel formulation proved efficient to provide the long-sought evidence that even doubly-deamidated Bcl-xL remains eligible for regulation by phosphorylation.

Bcl-xL deamidation and cancer: Charting the fame trajectories of legitimate child and hidden siblings.

Bcl-2 family proteins control programmed cell death through a complex network of interactions within and outside of this family, that are modulated by post-translational modifications (PTM). Bcl-xL, an anti-apoptotic member of this family, is overexpressed in a number of cancers, plays an important role in tumorigenesis and is correlated with drug resistance. Bcl-xL is susceptible to a number of different PTMs. Here, we focus on deamidation. We will first provide an overview of protein deamidation. We will then review how the apoptotic and autophagic functions of Bcl-xL are modified by this PTM, and how this impacts on its oncogenic properties. Possible therapeutic outcomes will also be discussed. Finally, we will highlight how the specific case of Bcl-xL deamidation provides groundings to revisit some concepts related to protein deamidation in general.

Synthetic toxic Aß1-42 oligomers can assemble in different morphologies.

BACKGROUND: Alzheimer's disease is the most common neurodegenerative disease associated with aggregation of Aß peptides. Aß toxicity is mostly related to the capacity of intermediate oligomers to disrupt membrane integrity. We previously expressed Aß1-42 in a eukaryotic cellular system and selected synthetic variants on their sole toxicity. The most toxic mutant G37C forms stable oligomers. METHODS: Different biophysical methods (Fluorescence spectroscopy, cross-linking, mass spectrometry (MS), Small Angle X-ray Scattering (SAXS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), calcein leakage) were used. RESULTS: The oligomers are mostly populated by a 14mers resulting from the packing of homodimers. These homodimers come from the formation of a disulfide bridge between two monomers. This link stabilizes the multimers and prevents the assembly into amyloid fibrils. These oligomers affect the membrane integrity. The reduction of disulfide bonds leads to a rearrangement and redirects assembly of Aß amyloid fibrils. CONCLUSION: The toxic synthetic AßG37C mutant can assemble into an amyloid of unusual morphology through the formation of anti-parallel ß-sheets. This pathway involves the formation of oligomers resulting from the arrangement of Aß dimers linked by covalent di-sulfide link, being these oligomers harmful for the membranes. GENERAL SIGNIFICANCE: The capacity to produce large amount of stable oligomers without additional detergents or extrinsic cross-linkers allow further structural and biophysical studies to understand their capacity to assemble and disrupt the membranes, a key event in Alzheimer's disease.

Combining native MS approaches to decipher archaeal box H/ACA ribonucleoprotein particle structure and activity.

Site-specific isomerization of uridines into pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.

Interaction of Aß(1-42) amyloids with lipids promotes "off-pathway" oligomerization and membrane damage.

The toxicity of amyloids, as Aß(1-42) involved in Alzheimer disease, is a subject under intense scrutiny. Many studies link their toxicity to the existence of various intermediate structures prior to fiber formation and/or their specific interaction with membranes. In this study we focused on the interaction between membrane models and Aß(1-42) peptides and variants (L34T, mG37C) produced in E. coli and purified in monomeric form. We evaluated the interaction of a toxic stable oligomeric form (oG37C) with membranes as comparison. Using various biophysical techniques as fluorescence and plasmon waveguide resonance, we clearly established that the oG37C interacts strongly with membranes leading to its disruption. All the studied peptides destabilized liposomes and accumulated slowly on the membrane (rate constant 0.02 min(-1)). Only the oG37C exhibited a particular pattern of interaction, comprising two steps: the initial binding followed by membrane reorganization. Cryo-TEM was used to visualize the peptide effect on liposome morphologies. Both oG37C and mG37C lead to PG membrane fragmentation. The PG membrane promotes peptide oligomerization, implicated in membrane disruption. WT (Aß(1-42)) also perturbs liposome organization with membrane deformation rather than disruption. For all the peptides studied, their interaction with the membranes changes their fibrillization process, with less fibers and more small aggregates being formed. These studies allowed to establish, a correlation between toxicity, fiber formation, and membrane disruption.

Assessment of human Nter and Cter BRCA1 mutations using growth and localization assays in yeast.

A large number of missense mutations have been identified within the tumor suppressor gene BRCA1. Most of them, called "variants of unknown significance" (VUS), cannot be classified as pathogenic or neutral by genetic methods, which complicates their cancer risk assessment. Functional assays have been developed to circumvent this uncertainty. They aim to determine how VUS impact the BRCA1 protein structure or function, thereby giving an indication of their potential to cause cancer. So far, three relevant assays have been designed in yeast and used on large sets of variants. However, they are limited to variants mapped in restricted domains of BRCA1. One of them, the small colony phenotype (SCP) assay, monitors the BRCA1-dependent growth of yeast colonies that increases with pathogenic but not neutral mutations positioned in the Cter region. Here, we extend this assay to the Nter part of BRCA1. We also designed a new assay, called the "yeast localization phenotype (YLP) assay," based on the accumulation of BRCA1 in a single inclusion body in the yeast nucleus. This phenotype is altered by variants positioned both in the Nter and Cter regions. Together, these assays provide new perspectives for the functional assessment of BRCA1 mutations in yeast.

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. We show that Tah1p can stabilize Pih1p in the absence of Hsp82 activity during the stationary phase of growth and consequently that the Tah1p:Pih1p interaction is sufficient for Pih1p stability. This prompted us to establish the solution structure of the Tah1p:Pih1p complex by NMR. The C-terminal tail S93-S111 of Tah1p snakes along Pih1p264-344 folded in a CS domain to form two intermolecular ß-sheets and one covering loop. However, a thorough inspection of the NMR and crystal structures revealed structural differences that may be of functional importance. In addition, our NMR and isothermal titration calorimetry data revealed the formation of direct contacts between Pih1p257-344 and the Hsp82MC domain in the presence of Tah1p. By co-expression in Escherichia coli, we demonstrate that Pih1p has two other direct partners, the Rsa1p assembly factor and the Nop58p core protein, and in vivo and in vitro experiments mapped the required binding domains. Our data suggest that these two interactions are mutually exclusive. The implication of this finding for box C/D small nucleolar ribonucleoparticle assembly is discussed.

A structure-toxicity study of Aß42 reveals a new anti-parallel aggregation pathway.

Amyloid beta (Aß) peptides produced by APP cleavage are central to the pathology of Alzheimer's disease. Despite widespread interest in this issue, the relationship between the auto-assembly and toxicity of these peptides remains controversial. One intriguing feature stems from their capacity to form anti-parallel ß-sheet oligomeric intermediates that can be converted into a parallel topology to allow the formation of protofibrillar and fibrillar Aß. Here, we present a novel approach to determining the molecular aspects of Aß assembly that is responsible for its in vivo toxicity. We selected Aß mutants with varying intracellular toxicities. In vitro, only toxic Aß (including wild-type Aß42) formed urea-resistant oligomers. These oligomers were able to assemble into fibrils that are rich in anti-parallel ß-sheet structures. Our results support the existence of a new pathway that depends on the folding capacity of Aß .

High-resolution structural analysis shows how Tah1 tethers Hsp90 to the R2TP complex.

The ubiquitous Hsp90 chaperone participates in snoRNP and RNA polymerase assembly through interaction with the R2TP complex. This complex includes the proteins Tah1, Pih1, Rvb1, and Rvb2. Tah1 bridges Hsp90 to R2TP. Its minimal TPR domain includes two TPR motifs and a capping helix. We established the high-resolution solution structures of Tah1 free and in complex with the Hsp90 C-terminal peptide. The TPR fold is similar in the free and bound forms and we show experimentally that in addition to its solvating/stabilizing role, the capping helix is essential for the recognition of the Hsp90 (704)EMEEVD(709) motif. In addition to Lys79 and Arg83 from the carboxylate clamp, this helix bears Tyr82 forming a π/S-CH3 interaction with Hsp90 M(705) from the peptide 310 helix. The Tah1 C-terminal region is unfolded, and we demonstrate that it is essential for the recruitment of the Pih1 C-terminal domain and folds upon binding.