scholarly journals The cryoEM structure of the fibril-forming low-complexity domain of hnRNPA2 reveals distinct differences from pathogenic amyloid and shows how mutation converts it to the pathogenic form

2020 ◽  
Author(s):  
Jiahui Lu ◽  
Qin Cao ◽  
Michael P. Hughes ◽  
Michael R. Sawaya ◽  
David R. Boyer ◽  
...  
Keyword(s):  
Low Complexity ◽  
Atomic Level ◽  
Missense Mutations ◽  
Cryo Electron Microscopy ◽  
Fibril Structure ◽  
Disease Mutations ◽  
Structural Differences ◽  
Energetic State ◽  
Protein Fibril

AbstracthnRNPA2 is one of a group of human ribonucleoproteins (RNPs) involved in RNA metabolism which form fibrils both under cellular stress and in mutated form in neurodegenerative conditions. Previous work established that the C-terminal low-complexity domain (LCD) of hnRNPA2 fibrillizes under stress, and that missense mutations in this domain are found in the disease multisystem proteinopathy (MSP) with symptoms indistinguishable from ALS and FTD. However, little is known at the atomic level about the hnRNPA2 LCD structure that is involved in those processes and how disease mutations cause structural change. Here we present the cryo-electron microscopy (cryoEM) structure of hnRNPA2 LCD fibril core and demonstrate its capability to form a reversible hydrogel in vitro containing amyloid-like fibrils. Whereas these fibrils, like pathogenic amyloid, are formed from protein chains stacked into β-sheets by backbone hydrogen bonds, they display distinct structural differences: the chains are kinked, enabling non-covalent cross-linking of fibrils and disfavoring formation of pathogenic steric zippers. Both their reversibility and energetic calculations suggest these fibrils are less stable than pathogenic amyloid. Moreover, the crystal structure of the disease-mutation-containing segment of hnRNPA2 suggests that the replacement fundamentally alters the fibril structure to a more stable energetic state. These findings illuminate how molecular interactions promote protein fibril networks and how mutation can transform fibril structure from functional to pathogenic form.

scholarly journals An empirical pipeline for personalized diagnosis of Lafora disease mutations

2021 ◽  
Author(s):  
M. Kathryn Brewer ◽  
Maria Machio-Castello ◽  
Rosa Viana ◽  
Jeremiah L Wayne ◽  
Andrea Kuchtova ◽  
...  
Keyword(s):  
Late Onset ◽  
Missense Mutations ◽  
Lafora Disease ◽  
Disease Mutations ◽  
Whole Exome ◽  
Functional Classes ◽  
Progressive Myoclonic Epilepsy ◽  
Personalized Diagnosis ◽  
Rapid Characterization

Lafora disease (LD) is a fatal, insidious metabolic disorder characterized by progressive myoclonic epilepsy manifesting in the teenage years, rapid neurological decline, and death typically within ten years of onset. Mutations in either EPM2A, encoding the glycogen phosphatase laforin, or EPM2B, encoding the E3 ligase malin, cause LD. Whole exome sequencing has revealed many EPM2A variants associated with late-onset or slower disease progression. We established an empirical pipeline for characterizing laforin missense mutations in vitro using complimentary biochemical approaches. Analysis of 26 mutations revealed distinct functional classes associated with different outcomes supported by multiple clinical cases. For example, F321C and G279C mutations have attenuated functional defects and are associated with slow progression. This pipeline allows rapid characterization and classification of novel EPM2A mutations, enabling clinicians and researchers to rapidly utilize genetic information to guide treatment of LD patients.

scholarly journals The CUL3–KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

2013 ◽  
Vol 451 (1) ◽  
pp. 111-122 ◽  
Author(s):  
Akihito Ohta ◽  
Frances-Rose Schumacher ◽  
Youcef Mehellou ◽  
Clare Johnson ◽  
Axel Knebel ◽  
...  
Keyword(s):  
Blood Pressure ◽  
E3 Ligase ◽  
Missense Mutations ◽  
Protein Levels ◽  
Disease Mutations ◽  
Cullin 3 ◽  
Interfering Rna ◽  
Ring E3 ◽  
Mutant Complex

The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

scholarly journals Structural differences between yeast and mammalian microtubules revealed by cryo-EM

2017 ◽  
Vol 216 (9) ◽  
pp. 2669-2677 ◽  
Author(s):  
Stuart C. Howes ◽  
Elisabeth A. Geyer ◽  
Benjamin LaFrance ◽  
Rui Zhang ◽  
Elizabeth H. Kellogg ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Size ◽  
Budding Yeast ◽  
Sequence Conservation ◽  
Gtp Hydrolysis ◽  
Allosteric Coupling ◽  
Cryo Electron Microscopy ◽  
Structural Differences

Microtubules are polymers of αβ-tubulin heterodimers essential for all eukaryotes. Despite sequence conservation, there are significant structural differences between microtubules assembled in vitro from mammalian or budding yeast tubulin. Yeast MTs were not observed to undergo compaction at the interdimer interface as seen for mammalian microtubules upon GTP hydrolysis. Lack of compaction might reflect slower GTP hydrolysis or a different degree of allosteric coupling in the lattice. The microtubule plus end–tracking protein Bim1 binds yeast microtubules both between αβ-tubulin heterodimers, as seen for other organisms, and within tubulin dimers, but binds mammalian tubulin only at interdimer contacts. At the concentrations used in cryo-electron microscopy, Bim1 causes the compaction of yeast microtubules and induces their rapid disassembly. Our studies demonstrate structural differences between yeast and mammalian microtubules that likely underlie their differing polymerization dynamics. These differences may reflect adaptations to the demands of different cell size or range of physiological growth temperatures.

scholarly journals α-Synuclein polymorphism determines oligodendroglial dysfunction

2021 ◽  
Author(s):  
Benedikt Frieg ◽  
James A Geraets ◽  
Timo Strohaeker ◽  
Christian Dienemann ◽  
Panagiota Mavroeidi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Brain Tissue ◽  
Ex Vivo ◽  
Neurological Diseases ◽  
Alpha Synuclein ◽  
Published Data ◽  
Cryo Electron Microscopy ◽  
Fibril Structure ◽  
Potential Biomarker

Synucleinopathies, such as Parkinson's disease (PD) and Multiple System Atrophy (MSA) are progressive and unremitting neurological diseases. For both PD and MSA, alpha-synuclein fibril inclusions inside brain cells are neuropathological hallmarks. In addition, amplification of alpha-synuclein fibrils from body fluids is a potential biomarker distinguishing PD from MSA. However, little is known about the structure of alpha-synuclein fibrils amplified from human samples and its connection to alpha-synuclein fibril structure in the human brain. Here we amplified alpha-synuclein fibrils from PD and MSA brain tissue, characterized its seeding potential in oligodendroglia, and determined the 3D structures by cryo-electron microscopy. We show that the alpha-synuclein fibrils from a MSA patient are more potent in recruiting the endogenous alpha-synuclein and evoking a redistribution of TPPP/p25alpha protein in mouse primary oligodendroglial cultures compared to those amplified from a PD patient. Cryo-electron microscopy shows that the PD- and MSA-amplified alpha-synuclein fibrils share a similar protofilament fold but differ in their inter-protofilament interface. The structures of the brain-tissue amplified alpha-synuclein fibrils are also similar to other in vitro and ex vivo alpha-synuclein fibrils. Together with published data, our results suggest that aSyn fibrils differ between PD and MSA in their quaternary arrangement and could further vary between different forms of PD and MSA.

Identification of new ARMC5 missense mutations in Primary Bilateral Macronodular Adrenal Hyperplasia (PBMAH) and their functional studies in vitro

2018 ◽  
Author(s):  
Anna Vaczlavik ◽  
Stephanie Espiard ◽  
Marie-Odile North ◽  
Ludivine Drougat ◽  
Marthe Rizk-Rabin ◽  
...  
Keyword(s):  
Missense Mutations ◽  
Adrenal Hyperplasia ◽  
Functional Studies

scholarly journals Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽  
2021 ◽  
Vol 11 (6) ◽  
pp. 915
Author(s):  
Jazelli Mueterthies ◽  
Davit A. Potoyan
Keyword(s):  
Phase Separation ◽  
Low Complexity ◽  
Nuclear Bodies ◽  
Disordered Proteins ◽  
Solvent Exposure ◽  
Ion Release ◽  
Dimer Formation ◽  
Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

scholarly journals Cryo-EM structure of amyloid fibrils formed by the entire low complexity domain of TDP-43

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qiuye Li ◽  
W. Michael Babinchak ◽  
Witold K. Surewicz
Keyword(s):  
Amyloid Fibrils ◽  
Low Complexity ◽  
Structural Features ◽  
Protein Fragments ◽  
Hydrophobic Residues ◽  
Backbone Conformation ◽  
Hydrophobic Amino Acids ◽  
Insight Into ◽  
Lateral Sclerosis

AbstractAmyotrophic lateral sclerosis and several other neurodegenerative diseases are associated with brain deposits of amyloid-like aggregates formed by the C-terminal fragments of TDP-43 that contain the low complexity domain of the protein. Here, we report the cryo-EM structure of amyloid formed from the entire TDP-43 low complexity domain in vitro at pH 4. This structure reveals single protofilament fibrils containing a large (139-residue), tightly packed core. While the C-terminal part of this core region is largely planar and characterized by a small proportion of hydrophobic amino acids, the N-terminal region contains numerous hydrophobic residues and has a non-planar backbone conformation, resulting in rugged surfaces of fibril ends. The structural features found in these fibrils differ from those previously found for fibrils generated from short protein fragments. The present atomic model for TDP-43 LCD fibrils provides insight into potential structural perturbations caused by phosphorylation and disease-related mutations.

scholarly journals Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

2005 ◽  
Vol 19 (7) ◽  
pp. 1884-1892 ◽  
Author(s):  
Helena Sim ◽  
Kieran Rimmer ◽  
Sabine Kelly ◽  
Louisa M. Ludbrook ◽  
Andrew H. A. Clayton ◽  
...  
Keyword(s):  
Y Chromosome ◽  
Nuclear Import ◽  
High Mobility ◽  
Sex Reversal ◽  
High Mobility Group ◽  
Missense Mutations ◽  
Nuclear Localization Signals ◽  
Xy Sex Reversal

Abstract The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-β binding to the C-terminal NLS (c-NLS), whereas in others, importin-β recognition is normal, suggesting the existence of an importin-β-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.

Abstract P459: Mavacamten And Danicamtiv Reverse Respective Contractile Abnormalities In Engineered Heart Tissue Models Of Hypertrophic And Dilated Cardiomyopathy

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Saiti S Halder ◽  
Lorenzo R Sewanan ◽  
Michael J Rynkiewicz ◽  
Jeffrey R Moore ◽  
William J Lehman ◽  
...  
Keyword(s):  
Dilated Cardiomyopathy ◽  
Calcium Sensitivity ◽  
Heart Tissue ◽  
Missense Mutations ◽  
Wild Type ◽  
Twitch Force ◽  
In Vitro Motility ◽  
Isometric Twitch ◽  
Engineered Heart Tissues

Missense mutations in alpha-tropomyosin (TPM1) can lead to development of hypertrophic (HCM) or dilated cardiomyopathy (DCM). HCM mutation E62Q and DCM mutation E54K have previously been studied extensively in experimental systems ranging from in vitro biochemical assays to animal models, although some conflicting results have been found. We undertook a detailed multi-scale assessment of these mutants that included atomistic simulations, regulated in vitro motility (IVM) assays, and finally physiologically relevant human engineered heart tissues. In IVM assays, E62Q previously has shown increased Calcium sensitivity. New molecular dynamics data shows mutation-induced changes to tropomyosin dynamics and interactions with actin and troponin. Human engineered heart tissues (EHT) were generated by seeding iPSC-derived cardiomyocytes engineered using CRISPR/CAS9 to express either E62Q or E54K cardiomyopathy mutations. After two weeks in culture, E62Q EHTs showed a drastically hypercontractile twitch force and significantly increased stiffness while displaying little difference in twitch kinetics compared to wild-type isogenic control EHTs. On the other hand, E54K EHTs displayed hypocontractile isometric twitch force with faster kinetics, impaired length-dependent activation and lowered stiffness. Given these contractile abnormalities, we hypothesized that small molecule myosin modulators to appropriately activate or inhibit myosin activity would restore E54K or E62Q EHTs to normal behavior. Accordingly, E62Q EHTs were treated with 0.5μM mavacamten (to remedy hypercontractility) and E54K EHTs with 0.5 μM danicamtiv (to remedy hypocontractility) for 4 days, followed by a 1 day washout period. Upon contractility testing, it was observed that the drugs were able to reverse contractile phenotypes observed in mutant EHTs and restore contractile properties to levels resembling those of the untreated wild type group. The computational, IVM and EHT studies provide clear evidence in support of the hyper- vs. hypo-contractility paradigm as a common axis that distinguishes HCM and DCM TPM1 mutations. Myosin modulators that directly compensate for underlying myofilament aberrations show promising efficacy in human in vitro systems.

Sign in / Sign up

Export Citation Format

Share Document