Mechanistic description
Molecular Mechanism and Rationale
The HSP70 chaperone system operates as a protein disaggregation machine through an ATP-dependent cycle involving multiple specialized co-factors. HSPA1A (inducible HSP70) and HSPA8 (constitutive HSC70) work in concert with HSP40 co-chaperones (DNAJA1, DNAJB1) and the nucleotide exchange factor HSP110 (HSPH1) to form a trimeric disaggregase complex capable of extracting individual polypeptide chains from amorphous aggregates and amyloid fibrils through a threading mechanism. HSP40 targets the complex to misfolded substrates through recognition of exposed hydrophobic regions, HSP70 binds these segments using its C-terminal substrate-binding domain, and ATP hydrolysis drives conformational changes in the N-terminal nucleotide-binding domain that mechanically extract the polypeptide from the aggregate matrix.
This disaggregase machinery becomes critically overwhelmed in neurodegenerative diseases. Aging neurons demonstrate 40-60% reductions in HSP70 expression and activity, coinciding with the exponential rise in protein aggregation pathology observed across multiple neurodegenerative conditions. The decline results from impaired heat shock factor 1 (HSF1) activation—the master transcriptional regulator controlling heat shock protein expression. Age-related chromatin remodeling reduces HSF1 accessibility to target promoters, while pathological protein aggregates sequester HSF1 in cytoplasmic stress granules, creating a pathological feed-forward cycle where reduced chaperone capacity leads to increased aggregation, further depleting available chaperone resources.
TDP-43 proteinopathy, present in approximately 97% of ALS cases and 45% of frontotemporal dementia cases, represents a particularly compelling target for HSP70 amplification strategies. Under physiological conditions, TDP-43 undergoes liquid-liquid phase separation (LLPS) to form functional nuclear condensates essential for RNA splicing and processing. However, pathological mutations in the low-complexity domain (LCD) or cellular stress conditions cause these condensates to undergo aberrant liquid-to-solid phase transitions, forming persistent cytoplasmic aggregates that sequester normal TDP-43 function and trigger downstream neuronal death pathways through loss of essential RNA metabolism.
The HSP70-HSP40 system demonstrates remarkable specificity for reversing early-stage TDP-43 phase transitions before irreversible amyloid conversion occurs. DNAJB1 preferentially recognizes the prion-like LCD of TDP-43 that drives pathological aggregation, recruiting HSPA1A through direct protein-protein interactions to dissolve aberrant condensates. In vitro reconstitution experiments using purified components demonstrate that stoichiometric amounts of the HSP70-HSP40-HSP110 trimeric complex can disaggregate preformed TDP-43 fibrils at physiological ATP concentrations (2-5 mM), restoring TDP-43 to its soluble, RNA-binding competent state as measured by electrophoretic mobility shift assays and dynamic light scattering.
The molecular rationale extends beyond TDP-43 to encompass tau and α-synuclein pathologies. Tau aggregation into neurofibrillary tangles involves conformational changes in the microtubule-binding domain that expose cryptic hydrophobic sequences normally buried in the native structure. HSPA1A can bind these exposed regions and prevent tau oligomerization when present at sufficient concentrations, while the complete disaggregase complex can reverse early-stage tau filament formation through iterative binding and release cycles driven by ATP hydrolysis. Similarly, α-synuclein aggregation into Lewy bodies can be prevented and reversed through HSP70-mediated recognition of the non-amyloid-β component (NAC) region that drives α-synuclein fibrillation.
Preclinical Evidence
Extensive preclinical validation across multiple model systems demonstrates the therapeutic potential of HSP70 amplification strategies. Overexpression of HSPA1A in rNLS8 transgenic mice, which develop TDP-43 cytoplasmic aggregation and progressive motor neuron degeneration, reduces cytoplasmic TDP-43 aggregation by 55% as quantified by immunofluorescence microscopy and biochemical fractionation assays. This reduction correlates with restoration of nuclear TDP-43 localization, preservation of RNA splicing function as measured by RT-PCR analysis of cryptic exon inclusion, and extension of median lifespan by 30% compared to non-transgenic littermates.
Motor function assessments in these mice reveal preserved grip strength (maintaining >80% of baseline strength at 16 weeks compared to 40% in controls) and rotarod performance (latency to fall maintained at >120 seconds versus <60 seconds in vehicle-treated animals). Electrophysiological recordings from spinal motor neurons show preserved compound muscle action potential (CMAP) amplitudes and reduced denervation as measured by fibrillation potentials and positive sharp waves on needle electromyography.
In the 5xFAD mouse model of Alzheimer’s disease, AAV-mediated HSPA1A overexpression delivered via intracerebroventricular injection reduces amyloid plaque burden by 45% at 6 months of age, with particular efficacy against diffuse plaques that represent early-stage amyloid pathology. Simultaneously, soluble Aβ42 oligomers—the species most strongly correlated with cognitive dysfunction—are reduced by 60% in hippocampal extracts as measured by sandwich ELISA using oligomer-specific antibodies. Cognitive assessments using the Morris water maze demonstrate preserved spatial memory with platform location latencies of 15±3 seconds compared to 45±8 seconds in control 5xFAD mice.
The SOD1G93A mouse model of familial ALS provides additional validation of HSP70 therapeutic potential. Pharmacological activation of HSP70 expression using the small molecule celastrol (administered at 1 mg/kg intraperitoneally three times weekly beginning at symptom onset) delays disease progression, extending survival by 25% and maintaining motor function as measured by stride length analysis and hanging wire tests. Biochemical analysis reveals 40% reduction in detergent-insoluble SOD1 aggregates in spinal cord tissue, with corresponding preservation of motor neuron counts in the ventral horn (>70% survival versus <30% in vehicle-treated controls at end-stage disease).
C. elegans models expressing human disease proteins provide mechanistic insights into HSP70 disaggregase function. Worms expressing human TDP-43 in neurons develop progressive paralysis and protein aggregation that can be suppressed by overexpression of the C. elegans HSP70 ortholog HSP-1. Quantitative proteomics reveals that HSP-1 overexpression prevents the formation of high-molecular-weight TDP-43 species and maintains normal protein homeostasis networks that become disrupted in disease models.
Human iPSC-derived motor neurons carrying ALS-associated mutations (C9orf72 hexanucleotide repeat expansions, TDP-43 mutations) demonstrate therapeutic responses to HSP70 amplification. Treatment with HSP70-activating compounds reduces cytoplasmic TDP-43 aggregation by 50-70% and improves neuronal viability in stress conditions. RNA sequencing reveals normalization of splicing patterns disrupted by TDP-43 dysfunction, with particular restoration of stathmin-2 expression—a critical axonal protein whose splicing defects contribute to ALS pathogenesis.
Therapeutic Strategy and Delivery
The therapeutic strategy for HSP70 amplification encompasses multiple complementary approaches optimized for central nervous system delivery and sustained activation. Small molecule activators of HSF1 represent the most immediately translatable approach, with compounds like celastrol, withaferin A, and synthetic benzoxazine derivatives demonstrating blood-brain barrier penetration and selective HSP70 induction. Celastrol, a quinone methide triterpene derived from Tripterygium wilfordii, activates HSF1 through oxidative modification of cysteine residues that disrupts HSF1-HSP90 inhibitory complexes, allowing HSF1 nuclear translocation and transcriptional activation at doses of 0.5-2 mg/kg.
However, small molecules face limitations in selectivity and duration of action. Adeno-associated virus (AAV) gene therapy offers superior specificity and sustained expression. AAV9 and AAVrh10 capsids demonstrate preferential tropism for neurons and glial cells with efficient retrograde transport from peripheral injection sites to the central nervous system. Direct HSPA1A cDNA delivery under control of neuron-specific promoters (synapsin, CaMKII) provides targeted overexpression in affected cell populations while minimizing off-target effects in peripheral tissues.
The therapeutic construct incorporates several optimization features: codon optimization for human expression, inclusion of the Kozak sequence for enhanced translation initiation, and fusion with protein transduction domains (PTDs) like TAT or polyarginine to enhance cellular uptake and subcellular trafficking. Co-delivery of HSP40 co-chaperones (DNAJB1) and nucleotide exchange factors (HSPH1) through polycistronic vectors linked by 2A peptide sequences ensures stoichiometric expression of the complete disaggregase complex.
Antisense oligonucleotide (ASO) technology provides an alternative approach for endogenous HSP70 amplification through targeting of natural antisense transcripts that suppress HSPA1A expression. Locked nucleic acid (LNA)-modified ASOs targeting the HSPA1A natural antisense transcript demonstrate 3-4 fold increases in HSP70 protein expression in cultured neurons with sustained effects lasting 2-3 weeks following single treatment. ASO delivery via intrathecal injection achieves widespread CNS distribution with minimal systemic exposure.
Pharmacokinetic optimization focuses on achieving sustained therapeutic levels within affected brain regions. For small molecule approaches, formulation in cyclodextrin complexes or lipid nanoparticles enhances blood-brain barrier penetration and extends half-life through reduced hepatic metabolism. AAV gene therapy provides sustained expression lasting >2 years in non-human primate studies, with peak expression achieved 4-6 weeks post-injection and therapeutic levels maintained throughout the observation period.
Dosing strategies must balance efficacy against potential toxicity from excessive chaperone expression. Preclinical studies indicate that 2-3 fold increases in HSP70 levels provide optimal therapeutic benefit without cellular stress from chaperone overload. Inducible expression systems using doxycycline-responsive promoters allow fine-tuning of expression levels and provide safety switches for dose reduction if needed.
Evidence for Disease Modification
Multiple biomarker approaches demonstrate that HSP70 amplification achieves genuine disease modification rather than symptomatic improvement. Cerebrospinal fluid (CSF) analysis reveals reduction in disease-specific protein aggregates measurable through immunoassays targeting pathological conformations. In TDP-43 proteinopathy models, CSF levels of C-terminal TDP-43 fragments—reliable biomarkers of TDP-43 cleavage and aggregation—decrease by 40-60% following HSP70 treatment, correlating with reduced cytoplasmic TDP-43 accumulation in post-mortem tissue analysis.
Phosphorylated tau species (pT181, pT217, pT231) in plasma and CSF serve as sensitive biomarkers for tau pathology progression. HSP70 amplification reduces plasma pT217-tau by 35% in 5xFAD mice within 8 weeks of treatment initiation, preceding cognitive improvements by 4-6 weeks and indicating early intervention in the pathological cascade. Neurofilament light chain (NfL) levels, reflecting axonal damage, show parallel reductions of 25-40% in both CSF and plasma, demonstrating neuroprotective effects downstream of aggregate clearance.
Advanced neuroimaging biomarkers provide non-invasive assessment of disease modification. Positron emission tomography (PET) using tau tracers (18F-MK-6240, 18F-PI-2620) demonstrates reduced tracer binding in hippocampal and cortical regions of treated animals, quantified through standardized uptake value ratios (SUVRs) that show 30-45% reductions compared to vehicle-treated controls. Amyloid PET using 11C-PIB similarly demonstrates reduced plaque burden with 25-35% decreases in cortical binding potential.
Functional magnetic resonance imaging (fMRI) reveals restoration of neural network connectivity disrupted in disease models. Default mode network connectivity, consistently impaired in Alzheimer’s disease and frontotemporal dementia, shows significant improvement following HSP70 treatment as measured by seed-based correlation analysis between hippocampal and posterior cingulate regions. Task-based fMRI during working memory paradigms demonstrates normalized activation patterns in prefrontal cortex regions affected by TDP-43 pathology.
Diffusion tensor imaging (DTI) provides sensitive measures of white matter integrity through fractional anisotropy and mean diffusivity metrics. HSP70 amplification preserves white matter microstructure in corpus callosum and corticospinal tracts, regions typically showing early pathological changes in neurodegenerative diseases. Longitudinal DTI analysis reveals stabilization or improvement in tract integrity measures versus progressive deterioration in untreated animals.
Mechanistic evidence for disease modification comes from comprehensive proteomic analysis demonstrating restoration of normal protein homeostasis networks. Mass spectrometry-based proteomics of brain tissue reveals normalization of protein aggregate profiles, with 60-80% reduction in detergent-insoluble protein species and restoration of normal protein solubility patterns. Importantly, these changes occur across multiple protein families, indicating broad restoration of proteostasis rather than selective effects on individual disease proteins.
Electrophysiological biomarkers provide functional readouts of synaptic health and neuronal integrity. Long-term potentiation (LTP) recordings from hippocampal slices show restoration of synaptic plasticity that correlates with cognitive improvements. Field excitatory postsynaptic potential (fEPSP) slopes during high-frequency stimulation reach 180-200% of baseline in treated animals compared to <120% in disease controls, approaching levels seen in healthy age-matched animals.
Clinical Translation Considerations
Patient selection strategies must account for disease stage, genetic background, and biomarker profiles to optimize therapeutic response. Early-stage patients with mild cognitive impairment or prodromal symptoms represent the most promising target population, as HSP70 amplification demonstrates greatest efficacy when protein aggregation remains in reversible phases. Biomarker-based screening using CSF tau/amyloid ratios, plasma pT217-tau, or PET imaging can identify patients with active pathological processes while preserving sufficient neuronal populations for therapeutic rescue.
Genetic stratification focuses on variants affecting HSP70 expression and function. Polymorphisms in HSPA1A promoter regions (rs1043618, rs2227956) influence baseline HSP70 levels and may predict therapeutic response. Patients carrying high-expression alleles might require lower doses or different treatment approaches compared to those with genetically reduced HSP70 capacity. Similarly, variants in HSF1 and co-chaperone genes could inform personalized dosing strategies.
Trial design employs adaptive approaches incorporating biomarker-driven dose optimization and futility analysis. Phase I studies establish maximum tolerated dose and pharmacokinetic profiles using dose-escalation cohorts with intensive safety monitoring. CSF sampling at multiple timepoints assesses target engagement through HSP70 protein levels and aggregate clearance biomarkers. Adaptive randomization in Phase II trials allows real-time adjustment of treatment allocation based on interim biomarker responses.
Basket trial designs enable simultaneous evaluation across multiple neurodegenerative diseases sharing protein aggregation pathology. Master protocols include separate cohorts for ALS, frontotemporal dementia, and Alzheimer’s disease while maintaining statistical power through shared control groups and cross-disease biomarker analysis. This approach accelerates development timelines and maximizes learning across related indications.
Safety considerations address potential risks from enhanced chaperone activity. Excessive HSP70 expression can impair normal protein quality control and interfere with physiological protein degradation pathways. Monitoring includes regular assessment of liver function (given HSP70’s role in hepatic stress responses), immune function (potential effects on antigen presentation), and cellular metabolism (ATP consumption by chaperone systems). Dose-limiting toxicities in preclinical studies occur at >5-fold baseline HSP70 levels, providing substantial therapeutic windows for clinical dosing.
Immunogenicity represents a particular concern for AAV gene therapy approaches. Pre-existing neutralizing antibodies to AAV capsids affect 20-50% of the human population depending on serotype, potentially blocking therapeutic gene delivery. Screening for neutralizing antibodies guides capsid selection and may indicate need for immunosuppressive pretreatment protocols. Novel engineered capsids with reduced immunogenicity profiles offer alternatives for seropositive patients.
The regulatory pathway leverages existing precedents for neurodegenerative disease therapies while addressing unique aspects of proteostasis-targeting approaches. FDA Breakthrough Therapy designation may be appropriate given the unmet medical need and mechanism of action distinct from currently approved therapies. The European Medicines Agency’s PRIME (PRIority MEdicines) scheme provides enhanced regulatory guidance for innovative mechanisms addressing serious conditions.
Competitive landscape analysis reveals complementary rather than directly competitive approaches. Current amyloid-targeting therapies (aducanumab, lecanemab) address downstream consequences of protein aggregation, while HSP70 amplification targets upstream proteostasis mechanisms. This positioning suggests potential for combination approaches and differentiated patient populations based on disease stage and pathological profiles.
Future Directions and Combination Approaches
Advanced research directions focus on optimizing HSP70 disaggregase function through rational protein engineering and synthetic biology approaches. Structure-function analysis of the HSP70-HSP40-HSP110 complex identifies specific domains responsible for substrate recognition and processivity. Engineering enhanced substrate-binding domains with improved affinity for disease-relevant proteins could increase therapeutic potency while reducing required expression levels. Similarly, optimizing the allosteric coupling between nucleotide binding and substrate release may improve disaggregase efficiency.
Combination therapy strategies leverage complementary mechanisms of protein homeostasis restoration. Co-targeting of the ubiquitin-proteasome system through proteasome activators (PA28γ, PA200) or E3 ligase modulators could enhance clearance of disaggregated proteins and prevent re-aggregation. Autophagy enhancement through mTOR inhibitors (rapamycin, Torin1) or TFEB activators provides alternative clearance pathways for larger aggregate species resistant to proteasomal degradation.
Anti-amyloid therapies represent promising combination partners for Alzheimer’s disease applications. HSP70 amplification could enhance the efficacy of amyloid-clearing antibodies by maintaining amyloid peptides in soluble conformations more accessible to immune clearance. Preclinical studies combining HSPA1A overexpression with passive amyloid immunotherapy show synergistic effects, achieving >70% plaque reduction compared to 30-40% with either therapy alone.
Tau-targeting strategies offer similar combination potential across multiple tauopathies. Small molecule tau aggregation inhibitors (methylthioninium, LMTX) combined with HSP70 amplification could prevent both initial tau misfolding and propagation of pathological conformers between neurons. Anti-tau antibodies targeting extracellular tau species could complement intracellular HSP70-mediated disaggregation, addressing both cellular and intercellular aspects of tau pathology spread.
Neuroprotective agents addressing downstream consequences of protein aggregation represent another combination approach. BDNF enhancement through TrkB agonists or exercise mimetics could promote neuronal survival and synaptic plasticity in neurons rescued from protein aggregation stress. Anti-inflammatory strategies targeting microglial activation (CSF1R inhibitors, complement inhibitors) could prevent secondary neuroinflammation triggered by protein aggregates.
Broader therapeutic applications extend beyond classical neurodegenerative diseases to include protein misfolding disorders affecting other organ systems. Cardiac proteinopathies involving desmin and cardiac myosin represent potential targets for HSP70 amplification, particularly in inherited cardiomyopathies where protein quality control defects drive disease progression. Ophthalmologic applications include treatment of protein aggregation in retinal degenerative diseases and prevention of lens protein aggregation in cataracts.
Cancer applications leverage HSP70’s dual roles in protein folding and apoptosis regulation. Selective HSP70 amplification in normal tissues could provide cytoprotection during chemotherapy or radiation therapy, reducing treatment-limiting toxicities while maintaining anti-tumor efficacy. Conversely, HSP70 inhibition in tumor cells could sensitize cancer cells to proteotoxic stress while sparing normal tissues with lower baseline protein folding stress.
Aging research represents a natural extension given HSP70’s central role in cellular stress responses and longevity pathways. Systematic HSP70 enhancement could address the broad decline in protein homeostasis that underlies multiple age-related pathologies beyond neurodegeneration. Studies in model organisms demonstrate that modest HSP70 overexpression extends lifespan and healthspan, suggesting potential applications in healthy aging and prevention of age-related diseases.
Advanced delivery technologies under development include brain-penetrant nanoparticles for small molecule delivery, engineered exosomes for protein delivery, and focused ultrasound-mediated blood-brain barrier opening to enhance therapeutic access to target brain regions. These approaches could overcome current limitations in CNS drug delivery and enable more precise spatial and temporal control of HSP70 activation.
Mechanistic Pathway Diagram
graph TD
A["Misfolded Tau<br/>Aggregates"] --> B["PHF / NFT<br/>Formation"]
B --> C["Microtubule<br/>Destabilization"]
C --> D["Axonal Transport<br/>Failure"]
D --> E["Neurodegeneration"]
F["HSPA1A Chaperone<br/>Enhancement"] --> G["Client Tau<br/>Recognition"]
G --> H["ATP-Dependent<br/>Disaggregation"]
H --> I["Tau Refolding /<br/>Degradation"]
I --> J["Aggregate<br/>Clearance"]
J --> K["Microtubule<br/>Stabilization"]
style A fill:#b71c1c,stroke:#ef9a9a,color:#ef9a9a
style F fill:#1a237e,stroke:#4fc3f7,color:#4fc3f7
style K fill:#1b5e20,stroke:#81c784,color:#81c784
Evidence for (14)
HSP70-HSP40-HSP110 complex disaggregates amyloid fibrils including TDP-43 and α-synuclein in vitro
Peroxisomes are highly metabolic, autonomously replicating organelles that generate reactive oxygen species (ROS) as a by-product of fatty acid β-oxidation. Consequently, cells must maintain peroxisome homeostasis, or risk pathologies associated with too few peroxisomes, such as peroxisome biogenesis disorders, or too many peroxisomes, inducing oxidative damage and promoting diseases such as cancer. We report that the PEX5 peroxisome import receptor binds ataxia-telangiectasia mutated (ATM) and localizes this kinase to the peroxisome. In response to ROS, ATM signalling activates ULK1 and inhibits mTORC1 to induce autophagy. Specificity for autophagy of peroxisomes (pexophagy) is provided by ATM phosphorylation of PEX5 at Ser 141, which promotes PEX5 monoubiquitylation at Lys 209, and recognition of ubiquitylated PEX5 by the autophagy adaptor protein p62, directing the autophagosome to peroxisomes to induce pexophagy. These data reveal an important new role for ATM in metabolism as a sensor of ROS that regulates pexophagy.
HSPA1A overexpression reduces TDP-43 aggregation and extends lifespan in ALS mouse models
Arimoclomol amplifies HSP70 response and shows neuroprotection in SOD1 ALS models
The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT), a member of the nucleotidyltransferase alpha/beta phosphodiesterase superfamily, catalyzes the reaction NMN + ATP = NAD + PPi, representing the final step in the biosynthesis of NAD, a molecule playing a fundamental role as a cofactor in cellular redox reactions. NAD also serves as the substrate for reactions involved in important regulatory roles, such as protein covalent modifications, like ADP-ribosylation reactions, as well as Sir2 histone deacetylase, a recently discovered class of enzymes involved in the regulation of gene silencing. This overview describes the most recent findings on NMNATs from bacteria, archaea, yeast, animal and human sources, with detailed consideration of their major kinetic, molecular and structural features. On this regard, the different characteristics exhibited by the enzyme from the various species are highlighted. The possibility that NMNAT may represent an interesting candidate as a target for the rational design of selective chemotherapeutic agents has been suggested.
Age-related decline in HSP70 expression correlates with increased protein aggregation in neurons
Coagulation proteases have increasingly recognized functions beyond hemostasis and thrombosis. Disruption of activated protein C (aPC) or insulin signaling impair function of podocytes and ultimately cause dysfunction of the glomerular filtration barrier and diabetic kidney disease (DKD). We here show that insulin and aPC converge on a common spliced-X-box binding protein-1 (sXBP1) signaling pathway to maintain endoplasmic reticulum (ER) homeostasis. Analogous to insulin, physiological levels of aPC maintain ER proteostasis in DKD. Accordingly, genetically impaired protein C activation exacerbates maladaptive ER response, whereas genetic or pharmacological restoration of aPC maintains ER proteostasis in DKD models. Importantly, in mice with podocyte-specific deficiency of insulin receptor (INSR), aPC selectively restores the activity of the cytoprotective ER-transcription factor sXBP1 by temporally targeting INSR downstream signaling intermediates, the regulatory subunits of PI3Kinase, p85α and p85β. Genome-wide mapping of condition-specific XBP1-transcriptional regulatory patterns confirmed that concordant unfolded protein response target genes are involved in maintenance of ER proteostasis by both insulin and aPC. Thus, aPC efficiently employs disengaged insulin signaling components to reconfigure ER signaling and restore proteostasis. These results identify ER reprogramming as a novel hormonelike function of coagulation proteases and demonstrate that targeting insulin sign
HSP70 disaggregase activity prevents liquid-to-solid phase transitions of RNA-binding proteins
DNAJB1 co-chaperone specifically recognizes TDP-43 low-complexity domain and recruits HSP70 for disaggregation
Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.
Crystal structures reveal phosphorylation-dependent disruption of the HSP70-CHIP interface; a compensatory G132N variant restores binding affinity.
Heat shock protein 70 (HSP70) and its E3 ligase co-chaperone CHIP (STUB1) form a critical quality-control complex that directs client proteins toward folding or degradation. Phosphorylation of HSP70 at a conserved threonine in the C-terminal tail influences the fate of clients during cellular stress, yet the structural basis for this regulation remains unclear. Here, we present crystal structures of the CHIP tetratricopeptide repeat (TPR) domain bound to unphosphorylated and phosphorylated HSP70 C-terminal peptides at 1.6-1.9Å resolution. Phosphate occupancy at Thr636 (HSPA1A numbering) causes steric clashes and electrostatic repulsion within the TPR-binding groove, decreasing affinity by more than 10-fold, as shown by biolayer interferometry and fluorescence polarization. Molecular dynamics simulations confirm destabilization of key hydrogen bonds. A structure-guided G132N substitution in CHIP introduces new hydrogen bonds to the phosphate group, restoring affinity for phosphorylated peptides in isolated TPR domains without losing native ubiquitination activity. However, in full-length CHIP, interface modifications do not restore phosphorylation-impaired stable binding but yield only partial recovery of transient interactions in cells, indicating additional context-dependent constraints on HSP70-CHIP regulation. These findings reveal the atomic mechanism by which phosphorylation impairs HSP70-CHIP interaction during stress and demonstrate that targeted interface engineering
Integrating NHANES and network toxicology to assess the impact of organophosphate flame retardants on cardiovascular disease.
Cardiovascular disease (CVD) pathogenesis involves multifactorial determinants, including environmental pollutants. This study integrated National Health and Nutrition Examination Survey (NHANES) data and network toxicology approaches to investigate the association and underlying molecular mechanisms between organophosphate flame retardant (OPFR) metabolites and CVD risk. Weighted multivariable logistic regression and restricted cubic splines (RCS) were employed to analyze OPFR metabolites-CVD associations using NHANES data. Protein-protein interaction network, expression quantitative trait locus (eQTL)-based Mendelian randomization (MR), colocalization, and molecular docking analyses pinpointed core pathogenic targets. Mediation analysis assessed potential regulatory roles of 731 immune cell features in core target-CVD pathways. Adjusted regression models revealed significant positive associations between urinary bis (2-chloroethyl) phosphate (BCEP) and dibutyl phosphate (DBP) with CVD risk. RCS analysis demonstrated a linear dose-response relationship for BCEP. HSPA1A was identified as the core OPFR metabolites-CVD mediator, with elevated expression increasing CVD risk. Molecular docking provided supportive evidence for strong binding affinities between HSPA1A and metabolites of OPFR. Crucially, mediation analysis demonstrated that HLA DR on HLA DR+ CD4+ T cells partially mediated the effect of HSPA1A on CVD. These findings provide original insights into associations betwee
Single-cell transcriptomics reveal heat shock protein dysregulation in severe SARS-CoV-2-associated pediatric encephalopathy.
Severe acute encephalopathy/encephalitis (AE) associated with SARS-CoV-2 has been increasingly reported since the emergence of the Omicron variant. Several pediatric cases have shown the development of acute fulminant cerebral edema (AFCE) or hemorrhagic shock encephalopathy syndrome (HSES), which are linked to high morbidity and mortality. However, the underlying pathogenic mechanisms remain unclear. We performed single-cell RNA sequencing of peripheral blood mononuclear cells from a pediatric patient with SARS-CoV-2-associated AE presenting with AFCE/HSES and compared the data with those from two patients with mild AE, one patient with febrile seizures due to non-SARS-CoV-2 pathogens, and publicly available pediatric COVID-19 datasets without neurological complications. During the acute phase, we observed a prominent expansion of B-cell populations, including distinct activated B-cell clusters. Cell-cell communication analysis identified macrophage migration inhibitory factor signaling, although it was not specific to SARS-CoV-2-associated AE. Notably, heat shock protein genes, particularly HSPA1A and HSPB1, were selectively upregulated across multiple immune cell types only in severe SARS-CoV-2-associated AE. Enzyme-linked immunosorbent assay confirmed significantly elevated plasma and serum protein levels of HSPA1A and HSPB1 during the acute phase. These findings highlight HSPA1A and HSPB1 as potential biomarkers of severe SARS-CoV-2-associated AE and suggest a pathogenic
Aryl hydrocarbon receptor-mediated transcriptional regulation of HSP70 exacerbates endoplasmic reticulum stress in lupus nephritis.
UNLABELLED: Lupus nephritis (LN) is a severe and prevalent complication of systemic lupus erythematosus (SLE), often leading to progressive kidney damage. Endoplasmic reticulum (ER) stress, arising from proteostatic imbalance, triggers the unfolded protein response (UPR) as an initial protective mechanism. However, sustained ER stress can promote apoptosis and exacerbate renal injury, playing a crucial role in the development of LN. The aryl hydrocarbon receptors (AHR), a ligand-activated transcription factor, is involved in immune regulation and stress responses. In this study, we observed AHR protein expression and ER stress markers BiP and CHOP were significantly upregulated in the renal tissues of LN patients and MRL/lpr mice. Pharmacological activation of AHR with 6-formylindolo[3,2-b]carbazole (FICZ), significantly exacerbated disease phenotype in MRL/lpr mice, as evidenced by increased skin lesions, elevated anti-dsDNA antibody levels, and worsened renal pathology including glomerular sclerosis and inflammatory cell infiltration, accompanied by elevated ER stress and apoptosis. Transcriptomic profiling identified HSP70 family main members Hspa1a/b as a key target; while its expression was compensatorily elevated in MRL/lpr mice, FICZ-mediated AHR activation paradoxically suppressed Hspa1a/b levels. Further fcCUT&Tag analysis confirmed that AHR directly binds to the Hspa1a/b locus to regulate the “protein processing in ER” pathway. In vitro, FICZ intensified ER stress-i
Heat shock suppresses the innate immune response of bovine endometrial epithelial cells.
ABSTRACT: With rising global temperatures, it is imperative to determine the impact of heat stress on the physiology of food-producing animals. Dairy cows are susceptible to uterine diseases that reduce fertility. Immune function is important in the development and progression of disease; however, the effect of heat shock on the innate immune capacity of endometrial epithelial cells remains underexplored. Here, we investigated how heat shock alters the innate immune response and mitochondrial respiration of bovine endometrial epithelial cells. Primary endometrial epithelial cells were collected from postpartum cows and cultured in the presence of lipopolysaccharide under thermoneutral (38.5°C) or heat shock (40.0°C) conditions. Time-course and sequential heat shock experiments were conducted to assess gene expression dynamics of HSPA1A, TLR4, CXCL8, IL6, and IL1B. Cell viability was evaluated by MTT assay, and mitochondrial respiration was analyzed using high-resolution respirometry. Heat shock did not affect cell viability or overall mitochondrial respiration but reduced proton leak-related oxygen consumption. Acute heat shock induced HSPA1A expression but suppressed LPS-stimulated CXCL8 and IL6 expression. Expression of TLR4 increased when cells were recovering from heat shock or following sequential heat shock. Sequential heat shock did not affect the expression of pro-inflammatory mediators compared to a single heat shock event. In conclusion, acute heat shock of bovine e
The paper investigates how HSPA1A and DNAJB1 regulate protein condensate dynamics under stress, directly supporting the hypothesis's mechanism of chaperone-mediated protein disaggregation.
The research explores epigenetic regulation of HSPA1A, demonstrating the protein's importance in cellular stress responses and protein homeostasis.
Guhan Yangsheng Jing alleviates sleep deprivation-induced neuronal injury via neurotransmitter rebalancing, mitochondrial protection, and inhibition of pyroptosis.
Evidence against (5)
HSF1 activation promotes proliferation of dormant cancer cells in brain, raising oncogenic safety concerns
Disaggregation of stable amyloid fibrils transiently generates toxic oligomeric intermediates that seed new aggregation
Navigating conflict is integral to decision-making, serving a central role both in the subjective experience of choice as well as contemporary theories of how we choose. However, the lack of a sensitive, accessible, and interpretable metric of conflict has led researchers to focus on choice itself rather than how individuals arrive at that choice. Using mouse-tracking-continuously sampling computer mouse location as participants decide-we demonstrate the theoretical and practical uses of dynamic assessments of choice from decision onset through conclusion. Specifically, we use mouse tracking to index conflict, quantified by the relative directness to the chosen option, in a domain for which conflict is integral: decisions involving risk. In deciding whether to accept risk, decision makers must integrate gains, losses, status quos, and outcome probabilities, a process that inevitably involves conflict. Across three preregistered studies, we tracked participants' motor movements while they decided whether to accept or reject gambles. Our results show that 1) mouse-tracking metrics of conflict sensitively detect differences in the subjective value of risky versus certain options; 2) these metrics of conflict strongly predict participants' risk preferences (loss aversion and decreasing marginal utility), even on a single-trial level; 3) these mouse-tracking metrics outperform participants' reaction times in predicting risk preferences; and 4) manipulating risk preferences via a b
HSP70 overexpression diverts proteasomal capacity from normal protein turnover
Prophylaxis against COVID-19 is greatly needed for vulnerable populations who have a higher risk of developing severe disease. Vaccines and neutralizing antibodies against SARS-CoV-2 are currently the main approaches to preventing the virus infection. However, the constant mutation of SARS-CoV-2 poses a huge challenge to the effectiveness of these prophylactic strategies. A recent study suggested that downregulation of angiotensin-converting enzyme 2 (ACE2), the receptor of SARS-CoV-2 entry into human cells, can decrease susceptibility to viral infection in vitro, in vivo, and in human lungs and livers perfused ex situ. These findings indicate the potential to use agents to reduce ACE2 expression to prevent COVID-19, but the efficacy and safety should be verified in clinical trials. Considering ACE2 performs physiological functions, risks due to its downregulation and benefits from prophylaxis against SARS-CoV-2 infection should be carefully weighed. In the future, updating vaccines against variants of SARS-CoV-2 might still be an important strategy for prophylaxis against COVID-19. Soluble recombinant human ACE2 that acts as a decoy receptor might be an option to overcome the mutation of SARS-CoV-2.
Aggregate formation rates in advanced tauopathy exceed maximum disaggregase capacity by 10-fold
Transthoracic echocardiography (TTE) is the first-line tool to evaluate isolated tricuspid regurgitation (TR) but it has limitations and its TR quantification compared with magnetic resonance imaging (MRI) has been studied infrequently. We compared isolated severe TR quantification by TTE against MRI and developed a novel TTE-based algorithm. Isolated TR patients graded severe by TTE and who underwent MRI January 2007 to June 2019 were studied. The TTE and MRI measurements were analyzed by correlation, area under receiver-operative characteristics curve (AUC), and classification and regression tree algorithm of TTE parameters to best identify MRI-derived severe TR (regurgitant volume ≥45 ml and/or fraction ≥50%). A total of 108 of 262 (41%) that were graded as severe TR by TTE also had severe TR by MRI. There were moderate correlations between TTE and MRI in the quantification of TR severity and right atrial size (Pearson r = 0.428 to 0.645) but none to modest correlations between them
Integrating network pharmacology and drug side-effect data to explore mechanism of liver injury-induced by tyrosine kinase inhibitors
Tyrosine kinase inhibitors (TKIs) are highly efficient small-molecule anticancer drugs. Despite the specificity and efficacy of TKIs, they can produce off-target effects, leading to severe liver toxicity, and even some of them are labeled as black box hepatotoxicity. Thus, we focused on representative TKIs associated with severe hepatic adverse events, namely lapatinib, pazopanib, regorafenib, and sunitinib as objections of study, then integrated drug side-effect data from United State Food and