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Post-translational modifications such as protein N-glycosylation, significantly influence cellular processes. Dysregulated N-glycosylation, exemplified in Grp94, a member of the Hsp90 family, leads to structural changes and the formation of epichaperomes, contributing to pathologies. Targeting N-glycosylation-induced conformations offers opportunities for developing selective chemical tools and drugs for these pathologic forms of chaperones. We here demonstrate how a specific Grp94 conformation induced by N-glycosylation, identified previously via molecular dynamics simulations, rationalizes the distinct behavior of similar ligands. Integrating dynamic ligand unbinding information with SAR development, we differentiate ligands productively engaging the pathologic Grp94 conformers from those that are not. Additionally, analyzing binding site stereoelectronic properties and QSAR models using cytotoxicity data unveils relationships between chemical, conformational properties, and biological activities. These findings facilitate the design of ligands targeting specific Grp94 conformations induced by abnormal glycosylation, selectively disrupting pathogenic protein networks while sparing normal mechanisms.
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Neurodegenerative disorders, including Alzheimer's disease (AD) and Parkinson's disease (PD), represent debilitating conditions with complex, poorly understood pathologies. Epichaperomes, pathologic protein assemblies nucleated on key chaperones, have emerged as critical players in the molecular dysfunction underlying these disorders. In this study, we introduce the synthesis and characterization of clickable epichaperome probes, PU-TCO, positive control, and PU-NTCO, negative control. Through comprehensive in vitro assays and cell-based investigations, we establish the specificity of the PU-TCO probe for epichaperomes. Furthermore, we demonstrate the efficacy of PU-TCO in detecting epichaperomes in brain tissue with a cellular resolution, underscoring its potential as a valuable tool for dissecting single-cell responses in neurodegenerative diseases. This clickable probe is therefore poised to address a critical need in the field, offering unprecedented precision and versatility in studying epichaperomes and opening avenues for novel insights into their role in disease pathology.
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The intricate protein-chaperone network is vital for cellular function. Recent discoveries have unveiled the existence of specialized chaperone complexes called epichaperomes, protein assemblies orchestrating the reconfiguration of protein-protein interaction networks, enhancing cellular adaptability and proliferation. This study delves into the structural and regulatory aspects of epichaperomes, with a particular emphasis on the significance of post-translational modifications in shaping their formation and function. A central finding of this investigation is the identification of specific PTMs on HSP90, particularly at residues Ser226 and Ser255 situated within an intrinsically disordered region, as critical determinants in epichaperome assembly. Our data demonstrate that the phosphorylation of these serine residues enhances HSP90's interaction with other chaperones and co-chaperones, creating a microenvironment conducive to epichaperome formation. Furthermore, this study establishes a direct link between epichaperome function and cellular physiology, especially in contexts where robust proliferation and adaptive behavior are essential, such as cancer and stem cell maintenance. These findings not only provide mechanistic insights but also hold promise for the development of novel therapeutic strategies targeting chaperone complexes in diseases characterized by epichaperome dysregulation, bridging the gap between fundamental research and precision medicine.
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Coherent spin dynamics of electrons in CdSe colloidal nanoplatelets are investigated by time-resolved pump-probe Faraday rotation at room and cryogenic temperatures. We measure electron spin precession in a magnetic field and determine g-factors of 1.83 and 1.72 at low temperatures for nanoplatelets with a thickness of 3 and 4 monolayers, respectively. The dephasing time of spin precession T2* amounts to a few nanoseconds and has a weak dependence on temperature, while the longitudinal spin relaxation time T1 exceeds 10 ns even at room temperature. Observations of single and double electron spin-flips confirm that the nanoplatelets are negatively charged. The spin-flip Raman scattering technique reveals g-factor anisotropy by up to 10% in nanoplatelets with thicknesses of 3, 4, and 5 monolayers. In the ensemble with a random orientation of nanoplatelets, our theoretical analysis shows that the measured Larmor precession frequency corresponds to the in-plane electron g-factor. We conclude that the experimentally observed electron spin dephasing and its acceleration in the magnetic field are not provided by the electron g-factor anisotropy and can be related to the localization of the resident electrons and fluctuations of the localization potential.
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Under normal conditions, heat shock proteins work in unison through dynamic protein interactions collectively referred to as the "chaperome." Recent work revealed that during cellular stress, the functional interactions of the chaperome are modified to form the "epichaperome," which results in improper protein folding, degradation, aggregation, and transport. This study is the first to investigate this novel mechanism of protein dishomeostasis in traumatic brain injury (TBI). Male and female adult, Sprague-Dawley rats received a lateral controlled cortical impact (CCI) and the ipsilateral hippocampus was collected 24 h 1, 2, and 4 weeks after injury. The epichaperome complex was visualized by measuring HSP90, HSC70 and HOP expression in native-PAGE and normalized to monomeric protein expression. A two-way ANOVA examined the effect of injury and sex at each time-point. Native HSP90, HSC70 and HOP protein expression showed a significant effect of injury effect across all time-points. Additionally, HSC70 and HOP showed significant sex effects at 24 h and 4 weeks. Altogether, controlled cortical impact significantly increased formation of the epichaperome across all proteins measured. Further investigation of this pathological mechanism can lead to a greater understanding of the link between TBI and increased risk of neurodegenerative disease and targeting the epichaperome for therapeutics.
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Lesiones Traumáticas del Encéfalo , Enfermedades Neurodegenerativas , Femenino , Masculino , Ratas , Animales , Ratas Sprague-Dawley , Análisis de Varianza , HipocampoRESUMEN
Optical alignment and optical orientation of excitons are studied experimentally on an ensemble of core/shell CdSe/CdS colloidal nanoplatelets. Linear and circular polarization of photoluminescence during resonant excitation of excitons is measured at cryogenic temperatures and with magnetic fields applied in the Faraday geometry. The developed theory addresses the optical alignment and optical orientation of excitons in colloidal nanocrystals, taking into account both bright and dark exciton states in the presence of strong electron-hole exchange interaction and the random in-plane orientation of nanoplatelets within the ensemble. Our theoretical analysis of the obtained experimental data allows us to evaluate the exciton fine structure parameters, the g-factors, and the spin lifetimes of the bright and dark excitons. The optical alignment effect enables the identification of the exciton and trion contributions to the emission spectrum, even in the absence of their clear separation in the spectra.
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Epichaperomes are disease-associated pathologic scaffolds, composed of tightly bound chaperones, co-chaperones, and other factors. They mediate anomalous protein-protein interactions inside cells, which aberrantly affects the function of protein networks, and in turn, cellular phenotypes. Epichaperome study necessitates the implementation of methods that retain these protein complexes in their native cellular states for analysis. Here we describe a protocol for detection and composition analysis of epichaperomes in cell homogenates through native polyacrylamide gel electrophoresis.
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Chaperonas Moleculares , Electroforesis en Gel de Poliacrilamida Nativa , Línea Celular , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de PoliacrilamidaRESUMEN
Systems-level assessments of protein-protein interaction (PPI) network dysfunctions are currently out-of-reach because approaches enabling proteome-wide identification, analysis, and modulation of context-specific PPI changes in native (unengineered) cells and tissues are lacking. Herein, we take advantage of chemical binders of maladaptive scaffolding structures termed epichaperomes and develop an epichaperome-based 'omics platform, epichaperomics, to identify PPI alterations in disease. We provide multiple lines of evidence, at both biochemical and functional levels, demonstrating the importance of these probes to identify and study PPI network dysfunctions and provide mechanistically and therapeutically relevant proteome-wide insights. As proof-of-principle, we derive systems-level insight into PPI dysfunctions of cancer cells which enabled the discovery of a context-dependent mechanism by which cancer cells enhance the fitness of mitotic protein networks. Importantly, our systems levels analyses support the use of epichaperome chemical binders as therapeutic strategies aimed at normalizing PPI networks.
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Neoplasias , Mapas de Interacción de Proteínas , Humanos , Proteoma/metabolismo , Mapeo de Interacción de Proteínas , Neoplasias/genética , AclimataciónRESUMEN
Protein-assembly defects due to an enrichment of aberrant conformational protein variants are emerging as a new frontier in therapeutics design. Understanding the structural elements that rewire the conformational dynamics of proteins and pathologically perturb functionally oriented ensembles is important for inhibitor development. Chaperones are hub proteins for the assembly of multiprotein complexes and an enrichment of aberrant conformers can affect the cellular proteome, and in turn, phenotypes. Here, we integrate computational and experimental tools to investigte how N-glycosylation of specific residues in glucose-regulated protein 94 (GRP94) modulates internal dynamics and alters the conformational fitness of regions fundamental for the interaction with ATP and synthetic ligands and impacts substructures important for the recognition of interacting proteins. N-glycosylation plays an active role in modulating the energy landscape of GRP94, and we provide support for leveraging the knowledge on distinct glycosylation variants to design molecules targeting GRP94 disease-associated conformational states and assemblies.
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Chaperonas Moleculares , Glicosilación , Ligandos , Chaperonas Moleculares/química , Conformación Proteica , Unión ProteicaRESUMEN
In this paper, we studied the role of the crystal structure in spheroidal CdSe nanocrystals on the band-edge exciton fine structure. Ensembles of zinc blende and wurtzite CdSe nanocrystals are investigated experimentally by two optical techniques: fluorescence line narrowing (FLN) and time-resolved photoluminescence. We argue that the zero-phonon line evaluated by the FLN technique gives the ensemble-averaged energy splitting between the lowest bright and dark exciton states, while the activation energy from the temperature-dependent photoluminescence decay is smaller and corresponds to the energy of an acoustic phonon. The energy splittings between the bright and dark exciton states determined using the FLN technique are found to be the same for zinc blende and wurtzite CdSe nanocrystals. Within the effective mass approximation, we develop a theoretical model considering the following factors: (i) influence of the nanocrystal shape on the bright-dark exciton splitting and the oscillator strength of the bright exciton, and (ii) shape dispersion in the ensemble of the nanocrystals. We show that these two factors result in similar calculated zero-phonon lines in zinc blende and wurtzite CdSe nanocrystals. The account of the nanocrystals shape dispersion allows us to evaluate the linewidth of the zero-phonon line.
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The coherent spin dynamics of electrons in CdSe nanocrystals embedded in a glass matrix with diameters from 3.3 up to 6.1 nm are investigated by time-resolved Faraday ellipticity at room and cryogenic temperatures. Only one Larmor precession frequency is detected, which corresponds to the larger of the two precession frequencies and thus g-factor values found in the typical signal from solution-grown colloidal CdSe nanocrystals. We identify this frequency accordingly as associated with the spin precession of resident electrons localized in the nanocrystals in the vicinity of the surface. We provide a detailed theoretical analysis of the exciton level spin structure in the magnetic field and model the spin dynamics in CdSe nanocrystals of different symmetries. This allows us to exclude the exciton as the origin of the experimentally observed oscillating signal. At a cryogenic temperature of 6 K, an additional nonoscillating component emerges in the spin dynamics. We consider several possible origins of this signal and conclude that it is related to the hole spin polarization.
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While initial theories on quantum confinement in colloidal quantum dots (QDs) led to analytical band gap/size relations or sizing functions, numerical methods describe size quantization more accurately. However, because of the lack of reliable sizing functions, researchers fit experimental band gap/size data sets using models with redundant, physically meaningless parameters that break down upon extrapolation. Here, we propose a new sizing function based on a proportional correction for nonparabolic bands. Using known bulk parameters, we predict size quantization for groups IV, III-V, II-VI, and IV-VI and metal-halide perovskite semiconductors, including straightforward adaptations for negative-gap semiconductors and nonspherical QDs. Refinement with respect to experimental data is possible using the Bohr diameter as a fitting parameter, by which we show a statistically relevant difference in the band gap/size relation for wurtzite and zinc blende CdSe. The general sizing function proposed here unifies the QD size calibration and enables researchers to assess bulk semiconductor parameters and predict the size quantization in unexplored materials.
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Cancer cell plasticity due to the dynamic architecture of interactome networks provides a vexing outlet for therapy evasion. Here, through chemical biology approaches for systems level exploration of protein connectivity changes applied to pancreatic cancer cell lines, patient biospecimens, and cell- and patient-derived xenografts in mice, we demonstrate interactomes can be re-engineered for vulnerability. By manipulating epichaperomes pharmacologically, we control and anticipate how thousands of proteins interact in real-time within tumours. Further, we can essentially force tumours into interactome hyperconnectivity and maximal protein-protein interaction capacity, a state whereby no rebound pathways can be deployed and where alternative signalling is supressed. This approach therefore primes interactomes to enhance vulnerability and improve treatment efficacy, enabling therapeutics with traditionally poor performance to become highly efficacious. These findings provide proof-of-principle for a paradigm to overcome drug resistance through pharmacologic manipulation of proteome-wide protein-protein interaction networks.
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Epigénesis Genética , Genoma , Chaperonas Moleculares/genética , Neoplasias/genética , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Animales , Femenino , Xenoinjertos , Humanos , Ratones , Transducción de SeñalRESUMEN
Diseases are a manifestation of how thousands of proteins interact. In several diseases, such as cancer and Alzheimer's disease, proteome-wide disturbances in protein-protein interactions are caused by alterations to chaperome scaffolds termed epichaperomes. Epichaperome-directed chemical probes may be useful for detecting and reversing defective chaperomes. Here we provide structural, biochemical, and functional insights into the discovery of epichaperome probes, with a focus on their use in central nervous system diseases. We demonstrate on-target activity and kinetic selectivity of a radiolabeled epichaperome probe in both cells and mice, together with a proof-of-principle in human patients in an exploratory single group assignment diagnostic study (ClinicalTrials.gov Identifier: NCT03371420). The clinical study is designed to determine the pharmacokinetic parameters and the incidence of adverse events in patients receiving a single microdose of the radiolabeled probe administered by intravenous injection. In sum, we introduce a discovery platform for brain-directed chemical probes that specifically modulate epichaperomes and provide proof-of-principle applications in their use in the detection, quantification, and modulation of the target in complex biological systems.
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Sistema Nervioso Central/metabolismo , Chaperonas Moleculares/metabolismo , Mapeo de Interacción de Proteínas/instrumentación , Proteoma/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Barrera Hematoencefálica/metabolismo , Neoplasias Encefálicas/diagnóstico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Supervivencia Celular/efectos de los fármacos , Sistema Nervioso Central/efectos de los fármacos , Glioblastoma/diagnóstico , Glioblastoma/metabolismo , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Ratones , Sondas Moleculares/química , Sondas Moleculares/farmacocinética , Sondas Moleculares/farmacología , Sondas Moleculares/uso terapéutico , Tomografía de Emisión de PositronesRESUMEN
The recombination dynamics and spin polarization of excitons in CdSe nanocrystals synthesized in a glass matrix are investigated using polarized photoluminescence in high magnetic fields up to 30 Tesla. The dynamics are accelerated by increasing temperature and magnetic field, confirming the dark exciton nature of low-temperature photoluminescence (PL). The circularly polarized PL in magnetic fields reveals several unusual appearances: (i) a spectral dependence of the polarization degree, (ii) its low saturation value, and (iii) a stronger intensity of the Zeeman component which is higher in energy. The latter feature is the most surprising being in contradiction with the thermal population of the exciton spin sublevels. The same contradiction was previously observed in the ensemble of wet-chemically synthesized CdSe nanocrystals but was not understood. We present a theory which explains all the observed features and shows that the inverted ordering of the circularly polarized PL maxima from the ensemble of nanocrystals is a result of competition between the zero phonon (ZPL) and one optical phonon-assisted (1PL) emission of the dark excitons. The essential aspects of the theoretical model are different polarization properties of the dark exciton emission via ZPL and 1PL recombination channels and the inhomogeneous broadening of the PL spectrum from the ensemble of nanocrystals exceeding the optical phonon energy.
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PURPOSE: Epichaperome network maintenance is vital to survival of tumors that express it. PU-H71 is an epichaperome inhibitor that binds to the ATP-binding site of HSP90 and has demonstrated antitumor activity in breast cancer xenograft models and clinical safety in patients. PU-positron emission tomography (PET) is a theragnostic imaging tool that allows visualization of the epichaperome target. In this phase Ib trial, we present safety and tolerability for PU-H71 plus nab-paclitaxel in HER2-negative patients with metastatic breast cancer (MBC) and the utility of PU-PET as a noninvasive predictive biomarker. METHODS: We performed a 3 + 3 dose-escalation study with escalating PU-H71 doses and standard nab-paclitaxel. The primary objective was to establish safety and determine maximum tolerated dose (MTD)/recommended phase 2 dose. Secondary objectives were to assess pharmacokinetics and clinical efficacy. Patients could enroll in a companion PU-PET protocol to measure epichaperome expression before treatment initiation to allow exploratory correlation with treatment benefit. RESULTS: Of the 12 patients enrolled, dose-limiting toxicity occurred in one patient (G3 neutropenic fever) at dose level 1; MTD of PU-H71 was 300 mg/m2 plus nab-paclitaxel 260 mg/m2 administered every 3 weeks. Common toxicities included diarrhea, fatigue, peripheral neuropathy, and nausea. PU-H71 systemic exposure was not altered by nab-paclitaxel administration. Two of 12 patients had partial response (overall response rate, 17%) and the clinical benefit rate was 42% (5 of 12). Time to progression was associated with baseline epichaperome positivity and PU-H71 peak standard uptake value (SUV), with more durable disease control observed with high epichaperome levels. CONCLUSION: The combination of PU-H71 and nab-paclitaxel was well tolerated, with evidence of clinical activity. More durable disease control without progression was observed in patients with high baseline epichaperome expression. A phase II trial of this combination with PU-PET as a companion diagnostic for patient selection is currently planned.
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Stresses associated with disease may pathologically remodel the proteome by both increasing interaction strength and altering interaction partners, resulting in proteome-wide connectivity dysfunctions. Chaperones play an important role in these alterations, but how these changes are executed remains largely unknown. Our study unveils a specific N-glycosylation pattern used by a chaperone, Glucose-regulated protein 94 (GRP94), to alter its conformational fitness and stabilize a state most permissive for stable interactions with proteins at the plasma membrane. This "protein assembly mutation' remodels protein networks and properties of the cell. We show in cells, human specimens, and mouse xenografts that proteome connectivity is restorable by inhibition of the N-glycosylated GRP94 variant. In summary, we provide biochemical evidence for stressor-induced chaperone-mediated protein mis-assemblies and demonstrate how these alterations are actionable in disease.
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Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Animales , Línea Celular Tumoral , Citosol/metabolismo , Glicosilación , Proteínas HSP70 de Choque Térmico/química , Humanos , Proteínas de la Membrana/química , Ratones Endogámicos NOD , Peso Molecular , Neoplasias/metabolismo , Oncogenes , Polisacáridos/metabolismo , Conformación ProteicaRESUMEN
Optimal functioning of neuronal networks is critical to the complex cognitive processes of memory and executive function that deteriorate in Alzheimer's disease (AD). Here we use cellular and animal models as well as human biospecimens to show that AD-related stressors mediate global disturbances in dynamic intra- and inter-neuronal networks through pathologic rewiring of the chaperome system into epichaperomes. These structures provide the backbone upon which proteome-wide connectivity, and in turn, protein networks become disturbed and ultimately dysfunctional. We introduce the term protein connectivity-based dysfunction (PCBD) to define this mechanism. Among most sensitive to PCBD are pathways with key roles in synaptic plasticity. We show at cellular and target organ levels that network connectivity and functional imbalances revert to normal levels upon epichaperome inhibition. In conclusion, we provide proof-of-principle to propose AD is a PCBDopathy, a disease of proteome-wide connectivity defects mediated by maladaptive epichaperomes.
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Enfermedad de Alzheimer/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Proteoma/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/patología , Mapeo Encefálico , Disfunción Cognitiva/metabolismo , Función Ejecutiva/fisiología , Femenino , Hipocampo/patología , Humanos , Masculino , Memoria/fisiología , Ratones , Vías NerviosasRESUMEN
The surface of nominally diamagnetic colloidal CdSe nanoplatelets can demonstrate paramagnetic behaviour owing to the uncompensated spins of dangling bonds, as we reveal here by optical spectroscopy in high magnetic fields up to 15 T using the exciton spin as a probe of the surface magnetism. The strongly nonlinear magnetic field dependence of the circular polarization of the exciton emission is determined by the magnetization of the dangling-bond spins (DBSs), the exciton spin polarization as well as the spin-dependent recombination of dark excitons. The sign of the exciton-DBS exchange interaction depends on the nanoplatelet growth conditions.