Oligomeric Protein Research Articles

Endothelial nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome regulation in atherosclerosis.

The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe-/- mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1β (IL-1β). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1β. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBβ to NF-κB Subunits.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical invivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor β (IκBβ). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBβ oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBβ oxomer formation did not occur via an IκBβ asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBβ ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBβ from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBβ contributes to the hypoxia-dependent regulation of inflammation.

Oligomerization of the IHF Protein in the Presence of Metal Cations

Oligomerization of the IHF Protein in the Presence of Metal Cations

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1β (IL-1β) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-β42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-β protein oligomers (AβO) (mainly AβO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aβ monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AβO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AβO42 specific thiolated-aptamer (AptAβO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AβO42 (MMGO-AuNPs-AptAβO42-AβO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AβO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 μL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AβO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Abstract 4376: The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer

Abstract Colorectal cancer (CRC) remains a major public health issue. Identification of biomarkers predicting high risk of recurrence and discovery of novel targets for therapy are urgently needed1. The cell surface receptor PTK7 is a poorly described evolutionary conserved member of the receptor tyrosine kinase superfamily that was first identified in human colon carcinoma and melanoma2-3. Accumulated data highlighted the implication of PTK7 in diverse cancer-related signaling pathways4-6 and, despite the lack of kinase activity, anti-PTK7 inhibitors are currently in development at preclinical and clinical stages7-9. The dual aim of this project is to increase the basic knowledge about PTK7 and to evaluate its role in CRC in order to propose in the future novel therapeutics directed against PTK7. With the aim to discover novel PTK7 associated pathways, we used a proximity biotinylation strategy coupled to mass spectrometry in CRC cells. Among the partners identified, we found that the pseudokinase PTK7 interacts with the active receptor tyrosine kinase EphrinA2 (EPHA2). Similarly to PTK7, EPHA2 expression correlates with poor prognosis in CRC10. In this work, we also demonstrate that PTK7 acts as a negative regulator of EPHA2 activation through the promotion of distinct oligomeric protein complexes and the modulation of EPHA2 phosphorylation. We also shed in light on the role of PTK7 in EPHA2 K63-linked ubiquitination and its subsequent endosomal sorting and lysosomal degradation. References 1 Punt CJ et al, Nat. Rev. Clin. Oncol., 2016; 2 Lhoumeau AC et al, Cell Cycle, 2011; 3 Lhoumeau AC et al, PLoS One, 2015; 4 Puppo F et al, EMBO reports, 2011; 5 Martinez S et al, J. Biol. Chem., 2015; 6 Daulat AM and Borg JP, Trends in Cancer, 2017; 7 Damelin M et al, Sci. Transl. Med., 2017; 8 Jie Y et al, Front. Immunol., 2021; 9 Maitland ML et al, Clin. Cancer. Res., 2021; 10 Dunne P et al, Clin. Cancer. Res., 2016; 11 Cioce M and Fazio VM, Cancers, 2021. Citation Format: Jean-Paul Borg, Charlotte Dessaux, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat. The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4376.

Structural Stabilization of Clinically Oriented Oligomeric Proteins During their Transit through Synthetic Secretory Amyloids.

Developing time-sustained drug delivery systems is a main goal in innovative medicines. Inspired by the architecture of secretory granules from the mammalian endocrine system it has generated non-toxic microscale amyloid materials through the coordination between divalent metals and poly-histidine stretches. Like their natural counterparts that keep the functionalities of the assembled protein, those synthetic structures release biologically active proteins during a slow self-disintegration process occurring in vitro and upon in vivo administration. Being these granules formed by a single pure protein species and therefore, chemically homogenous, they act as highly promising time-sustained drug delivery systems. Despite their enormous clinical potential, the nature of the clustering process and the quality of the released protein have been so far neglected issues. By using diverse polypeptide species and their protein-only oligomeric nanoscale versions as convenient models, a conformational rearrangement and a stabilization of the building blocks during their transit through the secretory granules, being the released material structurally distinguishable from the original source is proved here. This fact indicates a dynamic nature of secretory amyloids that act as conformational arrangers rather than as plain, inert protein-recruiting/protein-releasing granular depots.

Unraveling the impact of the p.R107L mutation on the structure and function of human αB-Crystallin: Implications for cataract formation

To date, several pathogenic mutations have been identified in the primary structure of human α-Crystallin, frequently involving the substitution of arginine with a different amino acid. These mutations can lead to the incidence of cataracts and myopathy. Recently, an important cataract-associated mutation has been reported in the functional α-Crystallin domain (ACD) of human αB-Crystallin protein, where arginine 107 (R107) is replaced by a leucine. In this study, we investigated the structure, chaperone function, stability, oligomerization, and amyloidogenic properties of the p.R107L human αB-Crystallin using a number of different techniques. Our results suggest that the p.R107L mutation can cause significant changes in the secondary, tertiary, and quaternary structures of αB-Crystallin. This cataractogenic mutation led to the formation of protein oligomers with larger sizes than the wild-type protein and reduced the chemical and thermal stability of the mutant chaperone. Both fluorescence and microscopic assessments indicated that this mutation significantly altered the amyloidogenic properties of human αB-Crystallin. Furthermore, the mutant protein indicated an attenuated in vitro chaperone activity. The molecular dynamics (MD) simulation confirmed the experimental results and indicated that p.R107L mutation could alter the proper conformation of human αB-Crystallin dimers. In summary, our results indicated that the p.R107L mutation could promote the formation of larger oligomers, diminish the stability and chaperone activity of human αB-Crystallin, and these changes, in turn, can play a crucial role in the development of cataract disorder.

Functional Selection of Tau Oligomerization‐Inhibiting Aptamers

AbstractPathological hyperphosphorylation and aggregation of microtubule‐associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next‐generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)‐induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3β‐mediated Tau hyperphosphorylation in cell‐free systems and okadaic acid‐mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro‐orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first‐in‐class neurotherapeutic to mitigate tauopathy‐associated neurodegenerative disorders.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3β-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Cysteine post-translational modifications regulate protein interactions of caveolin-3.

Caveolae are small flask-shaped invaginations of the surface membrane which are proposed to recruit and co-localize signaling molecules. The distinctive caveolar shape is achieved by the oligomeric structural protein caveolin, of which three isoforms exist. Aside from the finding that caveolin-3 is specifically expressed in muscle, functional differences between the caveolin isoforms have not been rigorously investigated. Caveolin-3 is relatively cysteine-rich compared to caveolins 1 and 2, so we investigated its cysteine post-translational modifications. We find that caveolin-3 is palmitoylated at 6 cysteines and becomes glutathiolated following redox stress. We map the caveolin-3 palmitoylation sites to a cluster of cysteines in its C terminal membrane domain, and the glutathiolation site to an N terminal cysteine close to the region of caveolin-3 proposed to engage in protein interactions. Glutathiolation abolishes caveolin-3 interaction with heterotrimeric G protein alpha subunits. Our results indicate that a caveolin-3 oligomer contains up to 66 palmitates, compared to up to 33 for caveolin-1. The additional palmitoylation sites in caveolin-3 therefore provide a mechanistic basis by which caveolae in smooth and striated muscle can possess unique phospholipid and protein cargoes. These unique adaptations of the muscle-specific caveolin isoform have important implications for caveolar assembly and signaling.

Thermally Reversible Gel-Sol Transition of Hydrogels via Dissociation and Association of an Artificial Protein Nanocage.

Oligomeric protein nanocages often disassemble into their subunits and reassemble by external stimuli. Thus, using these nanocages as cross-linkers for hydrogel network structures is a promising approach to allow hydrogels to undergo stimuli-responsive gel-sol transitions or self-healing. Here, we report hydrogels that show a reversible gel-sol transition resulting from the heat-induced dissociation and reassociation of protein nanocages. The hydrogel contained the 60-mer artificial protein nanocage, TIP60, as a supramolecular cross-linker for polyethylene glycol network structures. The hydrogel showed a gel-to-sol transition upon heating at a temperature above the melting point of TIP60 and immediately returned to a gel state upon cooling to room temperature. During the heating and cooling treatment of the hydrogel, small-angle X-ray scattering analysis suggested the dissociation and reassociation of TIP60. Furthermore, we demonstrated redox-responsive cargo release from TIP60 in the hydrogel. These results showed the potential of TIP60 as a component of multi-stimuli-responsive hydrogels.

Mechanistic insights into the role of amyloid-β in innate immunity

Colocalization of microbial pathogens and the β-amyloid peptide (Aβ) in the brain of Alzheimer’s disease (AD) patients suggests that microbial infection may play a role in sporadic AD. Aβ exhibits antimicrobial activity against numerous pathogens, supporting a potential role for Aβ in the innate immune response. While mammalian amyloid is associated with disease, many bacteria form amyloid fibrils to fortify the biofilm that protects the cells from the surrounding environment. In the microbial AD hypothesis, Aβ aggregates in response to infection to combat the pathogen. We hypothesize that this occurs through toxic Aβ oligomers that contain α-sheet structure and form prior to fibrillization. De novo designed α-sheet peptides specifically bind to the α-sheet structure present in the oligomers of both bacterial and mammalian amyloidogenic proteins to neutralize toxicity and inhibit aggregation. Here, we measure the effect of E. coli on Aβ, including upregulation, aggregation, and toxicity. Additionally, we determined the effect of Aβ structure on E. coli amyloid fibrils, or curli comprised of the CsgA protein, and biofilm formation. We found that curli formation by E. coli increased Aβ oligomer production, and Aβ oligomers inhibited curli biogenesis and reduced biofilm cell density. Further, curli and biofilm inhibition by Aβ oligomers increased E. coli susceptibility to gentamicin. Toxic oligomers of Aβ and CsgA interact via α-sheet interactions, neutralizing their toxicity. These results suggest that exposure to toxic oligomers formed by microbial pathogens triggers Aβ oligomer upregulation and aggregation to combat infection via selective interactions between α-sheet oligomers to neutralize toxicity of both species with subsequent inhibition of fibrillization.

Multivalent, calcium-independent binding of surfactant protein A and D to sulfated glycosaminoglycans of the alveolar epithelial glycocalyx.

Lung surfactant collectins, surfactant protein A (SP-A) and D (SP-D), are oligomeric C-type lectins involved in lung immunity. Through their carbohydrate recognition domain, they recognize carbohydrates at pathogen surfaces and initiate lung innate immune response. Here, we propose that they may also be able to bind to other carbohydrates present in typical cell surfaces, such as the alveolar epithelial glycocalyx. To test this hypothesis, we analyzed and quantified the binding affinity of SP-A and SP-D to different sugars and glycosaminoglycans (GAGs) by microscale thermophoresis (MST). In addition, by changing the calcium concentration, we aimed to characterize any consequences on the binding behavior. Our results show that both oligomeric proteins bind with high affinity (in nanomolar range) to GAGs, such as hyaluronan (HA), heparan sulfate (HS) and chondroitin sulfate (CS). Binding to HS and CS was calcium-independent, as it was not affected by changing calcium concentration in the buffer. Quantification of GAGs in bronchoalveolar lavage (BAL) fluid from animals deficient in either SP-A or SP-D showed changes in GAG composition, and electron micrographs showed differences in alveolar glycocalyx ultrastructure in vivo. Taken together, SP-A and SP-D bind to model sulfated glycosaminoglycans of the alveolar epithelial glycocalyx in a multivalent and calcium-independent way. These findings provide a potential mechanism for SP-A and SP-D as an integral part of the alveolar epithelial glycocalyx binding and interconnecting free GAGs, proteoglycans, and other glycans in glycoproteins, which may influence glycocalyx composition and structure.NEW & NOTEWORTHY SP-A and SP-D function has been related to innate immunity of the lung based on their binding to sugar residues at pathogen surfaces. However, their function in the healthy alveolus was considered as limited to interaction with surfactant lipids. Here, we demonstrated that these proteins bind to glycosaminoglycans present at typical cell surfaces like the alveolar epithelial glycocalyx. We propose a model where these proteins play an important role in interconnecting alveolar epithelial glycocalyx components.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling.

Self-assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size-tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative 'α/α' and 'α/β' interfaces, while α/α dimerization of wild-type KRAS promotes the secondary α/β interaction sequentially. Mutation-based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real-time NMR demonstrated that higher-order oligomers retain higher numbers of active GTP-bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster-enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle-based formulations of stable protein oligomers with enhanced cooperativity.

Conditional Cooperativity in RAS Assembly Pathways on Nanodiscs and Altered GTPase Cycling

AbstractSelf‐assemblies (i.e., nanoclusters) of the RAS GTPase on the membrane act as scaffolds that activate downstream RAF kinases and drive MAPK signaling for cell proliferation and tumorigenesis. However, the mechanistic details of nanoclustering remain largely unknown. Here, size‐tunable nanodisc platforms and paramagnetic relaxation enhancement (PRE) analyses revealed the structural basis of the cooperative assembly processes of fully processed KRAS, mutated in a quarter of human cancers. The cooperativity is modulated by the mutation and nucleotide states of KRAS and the lipid composition of the membrane. Notably, the oncogenic mutants assemble in nonsequential pathways with two mutually cooperative ‘α/α’ and ‘α/β’ interfaces, while α/α dimerization of wild‐type KRAS promotes the secondary α/β interaction sequentially. Mutation‐based interface engineering was used to selectively trap the oligomeric intermediates of KRAS and probe their favorable interface interactions. Transiently exposed interfaces were available for the assembly. Real‐time NMR demonstrated that higher‐order oligomers retain higher numbers of active GTP‐bound protomers in KRAS GTPase cycling. These data provide a deeper understanding of the nanocluster‐enhanced signaling in response to the environment. Furthermore, our methodology is applicable to assemblies of many other membrane GTPases and lipid nanoparticle‐based formulations of stable protein oligomers with enhanced cooperativity.

Synthetic β-sheets mimicking fibrillar and oligomeric structures for evaluation of spectral X-ray scattering technique for biomarker quantification.

Archetypical cross-β spines sharpen the boundary between functional and pathological proteins including β-amyloid, tau, α-synuclein and transthyretin are linked to many debilitating human neurodegenerative and non-neurodegenerative amyloidoses. An increased focus on development of pathogenic β-sheet specific fluid and imaging structural biomarkers and conformation-specific monoclonal antibodies in targeted therapies has been recently observed. Identification and quantification of pathogenic oligomers remain challenging for existing neuroimaging modalities. We propose two artificial β-sheets which can mimic the nanoscopic structural characteristics of pathogenic oligomers and fibrils for evaluating the performance of a label free, X-ray based biomarker detection and quantification technique. Highly similar structure with elliptical cross-section and parallel cross-β motif is observed among recombinant α-synuclein fibril, Aβ-42 fibril and artificial β-sheet fibrils. We then use these β-sheet models to assess the performance of spectral small angle X-ray scattering (sSAXS) technique for detecting β-sheet structures. sSAXS showed quantitatively accurate detection of antiparallel, cross-β artificial oligomers from a tissue mimicking environment and significant distinction between different oligomer packing densities such as diffuse and dense packings. The proposed synthetic β-sheet models mimicked the nanoscopic structural characteristics of β-sheets of fibrillar and oligomeric states of Aβ and α-synuclein based on the ATR-FTIR and SAXS data. The tunability of β-sheet proportions and shapes of structural motifs, and the low-cost of these β-sheet models can become useful test materials for evaluating β-sheet or amyloid specific biomarkers in a wide range of neurological diseases. By using the proposed synthetic β-sheet models, our study indicates that the sSAXS has potential to evaluate different stages of β-sheet-enriched structures including oligomers of pathogenic proteins.

Structural basis for regulated assembly of the mitochondrial fission GTPase Drp1

Mitochondrial fission is a critical cellular event to maintain organelle function. This multistep process is initiated by the enhanced recruitment and oligomerization of dynamin-related protein 1 (Drp1) at the surface of mitochondria. As such, Drp1 is essential for inducing mitochondrial division in mammalian cells, and homologous proteins are found in all eukaryotes. As a member of the dynamin superfamily of proteins (DSPs), controlled Drp1 self-assembly into large helical polymers stimulates its GTPase activity to promote membrane constriction. Still, little is known about the mechanisms that regulate correct spatial and temporal assembly of the fission machinery. Here we present a cryo-EM structure of a full-length Drp1 dimer in an auto-inhibited state. This dimer reveals two key conformational rearrangements that must be unlocked through intramolecular rearrangements to achieve the assembly-competent state observed in previous structures. This structural insight provides understanding into the mechanism for regulated self-assembly of the mitochondrial fission machinery.