Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Hydrogen deuterium uptake comparison between wild-type, I499F, and D380A variants of the myocilin olfactomedin domain

ABSTRACT: Studies of folded-to-misfolded transitions using model protein systems reveal a range of unfolding needed for exposure of amyloid-prone regions for subsequent fibrillization. Here, we probe the relationship between unfolding and aggregation for glaucoma-associated myocilin. Mutations within the olfactomedin domain of myocilin (OLF) cause a gain-of-function, namely cytotoxic intracellular aggregation, which hastens disease progression. Aggregation by wild-type OLF (OLFWT) competes with its chemical unfolding, but only below the threshold where OLF loses tertiary structure. Representative moderate (OLFD380A) and severe (OLFI499F) disease variants aggregate differently, with rates comparable to OLFWT in initial stages of unfolding, and variants adopt distinct partially folded structures seen along the OLFWT urea-unfolding pathway. Whether initiated with mutation or chemical perturbation, unfolding propagates outward to the propeller surface. In sum, for this large protein prone to amyloid formation, the requirement for a conformational change to promote amyloid fibrillization leads to direct competition between unfolding and aggregation.

INSTRUMENT(S): ACQUITY UPLC

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Minh Thu Ma

LAB HEAD: Raquel L Lieberman

PROVIDER: PXD045520 | Pride | 2023-11-27

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	D380A_OLF.DnX	Other
	D380A_OLF_cluster_data.csv	Csv
	D380A_OLF_relative_uptake_plots.zip	Other
	D380A_OLF_sequence.txt	Txt
	D380A_OLF_state_data.csv	Csv

Items per page:

1 - 5 of 18

Similar Datasets

Role of Complementarity-Determining Regions 1 and 3 in Pathologic Amyloid Formation by Human Immunoglobulin κ1 Light Chains

Project description:Immunoglobulin light chain (LC) amyloidosis is a life-threatening disease whose understanding and treatment is complicated by vast numbers of patient-specific mutations. To address molecular origins of the disease, we explored 14 patient-derived and engineered proteins related to κ1-family germline genes IGKVLD-33*01 and IGKVLD-39*01. Hydrogen-deuterium exchange mass spectrometry analysis of local conformational dynamics in full-length recombinant LCs and their fragments was integrated with studies of thermal stability, proteolytic susceptibility, amyloid formation, and amyloidogenic sequence propensities using spectroscopic, electron microscopic and bioinformatics tools. The results were mapped on the atomic structures of native and fibrillary proteins. Proteins from two κ1 subfamilies showed unexpected differences. Compared to their germline counterparts, amyloid LC related to IGKVLD-33*01 was less stable and formed amyloid faster, whereas amyloid LC related to IGKVLD-39*01 had similar stability and formed amyloid slower. These and other differences suggest different major factors influencing amyloid formation. In 33*01-related amyloid LC, these factors involved mutation-induced destabilization of the native structure and probable stabilization of amyloid. The atypical behavior of 39*01-related amyloid LC tracked back to increased dynamics/exposure of amyloidogenic segments in βC’V and βEV that could initiate aggregation, combined with decreased dynamics/exposure near the C23-Cys88 disulfide whose rearrangement is rate-limiting to amyloidogenesis. The results suggest distinct amyloidogenic pathways for closely related LCs and point to the antigen-binding regions CDR1 and CDR3, which are linked via the conserved internal disulfide, as key factors in amyloid formation by various LCs.

2023-05-23 | PXD039682 | Pride

Translocation of polyubiquitinated protein substrates by the hexameric Cdc48 ATPase

Project description:The hexameric Cdc48 ATPase (p97 or VCP in mammals) cooperates with its cofactor Ufd1/Npl4 to extract polyubiquitinated proteins from membranes or macromolecular complexes for degradation by the proteasome. Here, we clarify how the Cdc48 complex unfolds its substrates and translocates polypeptides with branchpoints. The Cdc48 complex recognizes primarily polyubiquitin chains, rather than the attached substrate. Cdc48 and Ufd1/Npl4 cooperatively bind the polyubiquitin chain, resulting in the unfolding of one ubiquitin molecule (initiator). Next, the ATPase pulls on the initiator ubiquitin and moves all ubiquitin molecules linked to its C-terminus through the central pore of the hexameric double-ring, causing transient ubiquitin unfolding. When the ATPase reaches the isopeptide bond of the substrate, it can translocate and unfold both N- and C-terminal segments. Ubiquitins linked to the branchpoint of the initiator dissociate from Ufd1/Npl4 and move outside the central pore, resulting in the release of unfolded, polyubiquitinated substrate from Cdc48.

2022-01-19 | PXD027639 | Pride

RNA-seq of mouse embryonic fibroblasts(MEFs) with knock-out of SERF2 compared to control MEFs

Project description:Serf2 has been identified as a potential modifier of aggregation in the case of several disease-related aggregation-prone proteins, such as amyloid-beta, alpha-synuclein and huntingtin. However, it's endogenous biological function remains unknown. Therefore, we generated Serf2-/- mice using the cre-lox system to explore the endogenous role of Serf2. As the knockout of Serf2 appeared to cause perinatal lethality, we isolated embryonic fibroblasts at embryonic day 13.5 from Serf2+/+ and Serf2-/- embryo's and generated monoclonal cell lines. We compared these cell lines through RNA-sequencing to investigate the role of Serf2 in embryonic development.

2023-01-08 | E-MTAB-10083 | biostudies-arrayexpress

Folding of Prestin’s Anion-Binding Site and the Mechanism of Outer Hair Cell Electromotility

Project description:Prestin responds to transmembrane voltage fluctuations by changing its cross-sectional area, a process underlying the electromotility of outer hair cells and cochlear amplification. Prestin belongs to the SLC26 family of anion transporters yet is the only member capable of displaying electromotility. Prestin’s voltage-dependent conformational changes are driven by the putative displacement of residue R399 and a set of sparse charged residues within the transmembrane domain, following the binding of a Cl- anion at a conserved binding site formed by amino termini of the TM3 and TM10 helices. However, a major conundrum arises as to how an anion that binds in proximity to a positive charge (R399), can promote the voltage sensitivity of prestin. Using hydrogen-deuterium exchange mass spectrometry, we find that prestin displays an unstable anion-binding site, where folding of the amino termini of TM3 and TM10 is coupled to Cl- binding. This event shortens the TM3-TM10 electrostatic gap, thereby connecting the two helices, resulting in reduced cross-sectional area. These folding events upon anion-binding are absent in SLC26A9, a non-electromotile transporter closely related to prestin. Dynamics of prestin embedded in a lipid bilayer closely match that in detergent micelle, except for a destabilized lipid-facing helix TM6 that is critical to prestin’s mechanical expansion. We observe helix fraying at prestin’s anion-binding site but cooperative unfolding of multiple lipid-facing helices, features that may promote prestin’s fast electromechanical rearrangements. These results highlight a novel role of the folding equilibrium of the anion-binding site, and helps define prestin’s unique voltage-sensing mechanism and electromotility.

2023-12-06 | PXD046965 | Pride

A conservative point mutation in a dynamic antigen-binding loop of human immunoglobulin λ6 light chain promotes pathologic amyloid formation

Project description:Immunoglobulin light chain (LC) amyloidosis (AL) is a life-threatening human disease wherein free monoclonal LCs deposit in vital organs. To determine what makes some LCs amyloidogenic, we explored patient-based amyloidogenic and non-amyloidogenic recombinant LCs from the λ6 subtype prevalent in AL. Hydrogen-deuterium exchange mass spectrometry, structural stability, proteolysis, and amyloid growth studies revealed that the antigen-binding CDR1 loop is the least protected part in the variable domain of λ6 LC, particularly in the AL variant. N32T substitution in CRD1 is identified as a driver of amyloid formation. Substitution N32T increased the amyloidogenic propensity of CDR1 loop, decreased its protection in the native structure, and accelerated amyloid growth in the context of other AL substitutions. The destabilizing effects of N32T propagated across the molecule increasing its dynamics in regions ~30Å away from the substitution site. Such striking long-range effects of a conservative point substitution in a dynamic surface loop may be relevant to Ig function. Comparison of patient-derived and engineered proteins showed that N32T interactions with other substitution sites must contribute to amyloidosis. The results suggest that CDR1 is critical in amyloid formation by other λ6 LCs.

2021-11-25 | PXD026528 | Pride

Simple and Fast Maximally Deuterated Control (maxD) preparation for HDX MS Experiments

Project description:During the analysis steps of hydrogen deuterium exchange (HDX) mass spectrometry (MS) there is an unavoidable loss of deuterons, or back-exchange. Understanding back-exchange is necessary to correct for loss during analysis, to calculate the absolute amount of exchange, and to ensure that deuterium recovery is as high as possible during LC-MS. Back-exchange can be measured and corrected for using a maximally deuterated species (here called maxD) in which the protein is deuterated at all backbone amide positions and analyzed with the same buffer components, %D2O, quenching conditions, and LC-MS parameters used during the analysis of other labeled samples. Here we describe a robust and broadly applicable protocol, using denaturation followed by deuteration, to prepare a maxD control sample in ~40 minutes. The protocol was evaluated with a number of proteins that varied in both size and folded structure. The relative fractional uptake and level of back-exchange with this protocol were both equivalent to those obtained with a previous protocol that requires much more time or one requiring isolation of peptic peptides prior to deuteration. Placing strong denaturation first in the protocol allowed maximum deuteration in a short time (~10 mins) with equal or more deuteration found in other methods. The absence of high temperatures and low pH during the deuteration step limited protein aggregation. This high-performance, fast, and easy to perform protocol should enhance routine preparation of maxD controls for both beginners and for challenging proteins.

2022-07-08 | PXD032924 | Pride

Widespread Influence of 3′-end Structures on Mammalian mRNA Processing and Stability [Artificial-3'-end]

Project description:Understanding the physiological relevance of structures in mammalian mRNAs remains elusive, especially considering the global unfolding of mRNA structures in eukaryotic organisms recently examined, as well as the decade-long observation that mRNAs generally seem no more likely than random sequences to be stably folded. Here we show that RNA secondary structures, mostly weak and close-to-random, facilitate the 3′-end processing of thousands of human mRNAs by juxtaposing poly(A) signals (PASs) and cleavage sites that are otherwise too far apart. Folding of these 3′-end structures also enhances mRNA stability. Global structure probing shows that 3′-end regions are indeed folded in cells despite substantial unfolding of PAS-upstream regions. Analyses of thousands of ectopically expressed variants prove that folding both enhances processing and increases stability. Mutagenesis of a genomic locus further implicates structure-controlled processing in regulating neighboring gene expression. These results reveal widespread roles for RNA structure in mammalian mRNA biogenesis and metabolism.

2017-03-12 | GSE95467 | GEO

Widespread Influence of 3′-end Structures on Mammalian mRNA Processing and Stability [CENPB-3'-end-library]

2017-03-12 | GSE93424 | GEO