Search Results (2,027)

The rabies virus (RABV) phosphoprotein (P protein) has multiple functions, including acting as the essential non-catalytic cofactor of the viral polymerase (L protein) for genome replication and transcription; the principal viral antagonist of the interferon (IFN)-mediated innate immune response; and the chaperone for the viral nucleoprotein (N protein). Although P protein is known to undergo phosphorylation by cellular kinases, the location and functions of the phosphorylation sites remains poorly defined. Here, we report the identification by mass-spectrometry (MS) of residues of P protein that are modified by phosphorylation in mammalian cells, including several novel sites. Analysis of P protein with phospho-mimetic and phospho-inhibitory mutations of three novel residues/clusters that were commonly identified by MS (Ser48, Ser183/187, Ser217/219/220) indicate that phosphorylation at each of these sites does not have a major influence on nuclear trafficking or antagonistic functions toward IFN signalling pathways. However, phosphorylation of Ser48 in the N-terminus of P protein impaired function in transcription/replication and in the formation of replication structures that contain complexes of P and N proteins, suggestive of altered interactions of these proteins. The crystal structure of P protein containing the S48E phospho-mimetic mutation indicates that Ser48 phosphorylation facilitates the binding of residues 41–52 of P protein into the RNA-binding groove of non-RNA-bound N protein (N⁰), primarily through the formation of a salt bridge with Arg434 of N protein. These data indicate that Ser48 modification regulates the cycling of P-N⁰ chaperone complexes that deliver N protein to RNA to enable transcription/replication, such that enhanced interaction due to S48E phospho-mimetic mutation reduces N protein delivery to the RNA, inhibiting subsequent transcription/replication processes. These data are, to our knowledge, the first to implicate phosphorylation of RABV P protein in conserved replication functions of the P gene. Full article

Molecular chaperones, especially heat shock proteins (HSPs) have vital functions in cells’ responses to stress. Here, we cloned and sequenced the complete complementary DNA encoding HSP90 (MjHSP90) from the shrimp Marsupenaeus japonicus. The MjHSP90 cDNA comprised 3162 bp, including a 2172 bp coding region encoding a 724 amino acid-protein (predicted molecular mass = 83.12 kDa). Homology and phylogenetic analyses showed that MjHSP90 was highly conserved and most homologous to Litopenaeus vannamei HSP90. MjHSP90 is expressed in all tested tissues, with high expression in gill tissue and the hepatopancreas. Cold stress significantly upregulated MjHSP90 expression in the gill and hepatopancreas (p < 0.05). Following RNA interference knockdown of MjHSP90, the cold stress-related death rate of the shrimp increased significantly, accompanied by significantly upregulated expression of apoptosis-related genes Mjcaspase-3 and Mjbcl-2 (p < 0.05) and an increase in the number of apoptotic cells. The results indicated that MjHSP90 might play a pivotal role in the shrimp’s immune response to cold stress. Full article

Alzheimer’s disease (AD) is increasingly recognized as a multifactorial disorder driven by a combination of disruptions in proteostasis and organelle communication. The 2020 Lancet commission reported that approximately 10 million people worldwide were affected by AD in the mid-20th century. AD is the most prevalent cause of dementia. By early 2030, the global cost of dementia is projected to rise by USD 2 trillion per year, with up to 85% of that cost attributed to daily patient care. Several factors have been implicated in the progression of neurodegeneration, including increased oxidative stress, the accumulation of misfolded proteins, the formation of amyloid plaques and aggregates, the unfolded protein response (UPR), and mitochondrial–endoplasmic reticulum (ER) calcium homeostasis. However, the exact triggers that initiate these pathological processes remain unclear, in part because clinical symptoms often emerge gradually and subtly, complicating early diagnosis. Among the early hallmarks of neurodegeneration, elevated levels of reactive oxygen species (ROS) and the buildup of misfolded proteins are believed to play pivotal roles in disrupting proteostasis, leading to cognitive deficits and neuronal cell death. The accumulation of amyloid-β (Aβ) plaques and tau neurofibrillary tangles is a characteristic feature of AD. These features contribute to chronic neuroinflammation, which is marked by the release of pro-inflammatory cytokines and chemokines that exacerbate oxidative stress. Given these interconnected mechanisms, targeting stress-related signaling pathways, such as oxidative stress (ROS) generated in the mitochondria and ER, ER stress, UPR, and cytosolic chaperones, represents a promising strategy for therapeutic intervention. This review focuses on the relationship between stress chaperone responses and organelle function, particularly the interaction between mitochondria and the ER, in the development of new therapies for AD and related neurodegenerative disorders. Full article

RNA-seq analysis of the highly differentiated human retinal pigment epithelial (RPE) cell-line ARPE-19, cultured on transwells for ≥4 months, yielded 44,909 genes showing 83.35% alignment with the human reference genome. These included mRNA transcripts of RPE-specific genes and those involved in retinopathies. Monolayers were fed photoreceptor outer segments (POS), designed to be synchronously internalised, mimicking homeostatic RPE activity. Cells were subsequently fixed at 4, 6, 24 and 48 h when POS were previously shown to maximally co-localise with Rab5, Rab7, LAMP/lysosomes and LC3b/autophagic compartments. A comprehensive analysis of differentially expressed genes involved in proteolysis revealed a pattern of gene orchestration consistent with POS breakdown in the autophagy-lysosomal pathway. At 4 h, these included elevated upstream signalling events promoting early stages of cargo transport and endosome maturation compared to RPE without POS exposure. This transcriptional landscape altered from 6 h, transitioning to promoting cargo degradation in autolysosomes by 24–48 h. Longitudinal scrutiny of mRNA transcripts revealed nuanced differences even within linked gene networks. POS exposure also initiated transcriptional upregulation in ubiquitin proteasome and chaperone-mediated systems within 4–6 h, providing evidence of cross-talk with other proteolytic processes. These findings show detailed evidence of transcriptome-level responses to cargo trafficking and processing in RPE cells. Full article

The seasonal patterns of viral diseases in farm animals present significant challenges to global livestock productivity, with cold stress emerging as a potential modulator of host–pathogen interactions. This review synthesizes current knowledge on the expression dynamics of heat shock protein 70 (HSP70) in farm animals under cold-stress conditions and its potential roles as (1) a viral replication facilitator and (2) an immune response regulator. This review highlights cold-induced HSP70 overexpression in essential organs, as well as its effects on significant virus life cycles, such as porcine epidemic diarrhea virus (PEDV), porcine reproductive and respiratory syndrome virus (PRRSV), and bovine viral diarrhea virus (BVDV), through processes like viral protein chaperoning, replication complex stabilization, and host defense modulation. By integrating insights from thermophysiology, virology, and immunology, we suggest that HSP70 serves as a crucial link between environmental stress and viral disease seasonality. We also discuss translational opportunities targeting HSP70 pathways to break the cycle of seasonal outbreaks, while addressing key knowledge gaps requiring further investigation. This article provides a framework for understanding climate-driven disease patterns and developing seasonally adjusted intervention strategies. Full article

Protein degradation plays a fundamental role in maintaining protein homeostasis and ensures proper cellular function by regulating protein quality and quantity. Heat shock protein 100 (Hsp100), found in bacteria, plants, and fungi, is a unique chaperone family responsible for rescuing misfolded proteins from aggregated states in an ATP-dependent manner. To date, they are primarily known to mediate heat stress adaptation and enhance cellular survival under extreme conditions in higher plants and algae. Resting cyst formation in dinoflagellates is widely recognized as a response to adverse conditions, which offers an adaptive advantage to endure harsh environmental extremes that are unsuitable for vegetative cell growth and survival. In this study, based on a full-length cDNA sequence, we characterized an Hsp100 gene (SaHsp100) from the cosmopolitan bloom-forming dinoflagellate Scrippsiella acuminata, aiming to examine its life stage-specific expression patterns and preliminarily explore its potential functions. The qPCR results revealed that Hsp100 transcript levels were significantly elevated in newly formed resting cysts compared to vegetative cells and continued to increase during storage under simulated marine sediment conditions (darkness, low temperature, and anoxia). Parallel reaction monitoring (PRM)-based quantification further confirmed that Hsp100 protein levels were significantly higher in resting cysts than in vegetative cells and increased after three months of storage. These findings collectively highlighted the fundamental role of Hsp100 in the alteration of the life cycle and dormancy maintenance of S. acuminata, likely by enhancing stress adaptation and promoting cell survival through participation in proteostasis maintenance, particularly under natural sediment-like conditions that trigger severe abiotic stress. Our work deepens the current understanding of Hsp family members in dinoflagellates, paving the way for future investigations into their ecological relevance within this ecologically significant group. Full article

Heat shock protein 90 (HSP90) is a molecular chaperone that plays a pivotal role in the stabilization and functional activation of numerous oncoproteins and signaling molecules essential for cancer cell survival and proliferation. Despite the extensive development and clinical evaluation of HSP90 inhibitors, their therapeutic potential as monotherapies has been limited by suboptimal efficacy, dose-limiting toxicity, and the emergence of drug resistance. Recent studies have demonstrated that combination therapies involving HSP90 inhibitors and other anticancer agents such as chemotherapeutics, targeted therapies, and immune checkpoint inhibitors can enhance anticancer activity, overcome resistance mechanisms, and modulate the tumor microenvironment. These synergistic effects are mediated by the concurrent degradation of client proteins, the disruption of signaling pathways, and the enhancement of antitumor immunity. However, the successful clinical implementation of such combination strategies requires the careful optimization of dosage, administration schedules, toxicity management, and patient selection based on predictive biomarkers. In this review, we provide a comprehensive overview of the mechanistic rationale, preclinical and clinical evidence, and therapeutic challenges associated with HSP90 inhibitor-based combination therapies. We also discuss future directions leveraging emerging technologies including multi-omics profiling, artificial intelligence, and nanoparticle-mediated delivery for the development of personalized and effective combination regimens in oncology. Full article

Background/Objectives: The treatment of multiple myeloma (MM) remains a challenge, as almost all patients will eventually relapse. Proteasome inhibitors are a cornerstone in the management of MM. Unfortunately, validated biomarkers predicting drug response are largely missing. Therefore, we aimed to identify genes associated with drug resistance or sensitization to proteasome inhibitors. Methods: We performed genome-wide CRISPR-Cas9 knockout (KO) screens in human KMS-28-BM myeloma cells to identify genetic determinants associated with resistance or sensitization to proteasome inhibitors. Results: We show that KO of KLF13 and PSMC4 induces drug resistance, while NUDCD2, OSER1 and HERC1 KO cause drug sensitization. Subsequently, we focused on top sensitization hit, NUDCD2, which acts as a co-chaperone of Hsp90 to regulate the LIS1/dynein complex. RNA sequencing showed downregulation of genes involved in the ERAD pathway and in ER-associated ubiquitin-dependent protein catabolic processes in both untreated and carfilzomib-treated NUDCD2 KO cells, suggesting that NUDCD2 depletion alters protein degradation. Furthermore, bortezomib-treated NUDCD2 KO cells showed a decreased expression of genes that have a function in oxidative phosphorylation and the mitochondrial membrane, such as Carnitine Palmitoyltransferase 1A (CPT1A). CPT1A catalyzes the uptake of long chain fatty acids into mitochondria. Mitochondrial lipid metabolism has recently been reported as a possible therapeutic target for MM drug sensitivity. Conclusions: These results contribute to the search for therapeutic targets that can sensitize MM patients to proteasome inhibitors. Full article

Polyethylene terephthalate (PET) pollution represents a significant environmental challenge due to its widespread use and recalcitrant nature. PET-degrading enzymes, particularly Ideonella sakaiensis PETases (IsPETase), have emerged as promising biocatalysts for mitigating this problem. This review provides a comprehensive overview of recent advancements in the discovery and heterologous expression of IsPETase and closely related enzymes. We highlight innovative approaches, such as in silico and AI-based enzyme screening and advanced screening assays. Strategies to enhance enzyme secretion and solubility, such as using signal peptides, fusion tags, chaperone co-expression, cell surface display systems, and membrane permeability modulation, are critically evaluated. Despite considerable progress, challenges remain in achieving industrial-scale production and application. Future research must focus on integrating cutting-edge molecular biology techniques with host-specific optimisation to achieve sustainable and cost-effective solutions for PET biodegradation and recycling. This review aims to provide a foundation for further exploration and innovation in the field of enzymatic plastic degradation. Full article

Hyperekplexia (OMIM 149400), a sensorimotor syndrome of perinatal clinical relevance, causes newborns to display an energic startle reflex in response to certain trivial stimuli. This condition can be lethal due to apnea episodes. The disease is caused by a blockade of glycinergic neurotransmission. Glycinergic interneurons preserve their identity by the activity of the surface glycine transporter GlyT2, which supplies glycine to presynaptic terminals to maintain glycine content in synaptic vesicles. Loss-of-function mutations in the GlyT2 gene (SLC6A5) cause a presynaptic form of human hyperekplexia. Here, we describe a new GlyT2 variant found in an infantile patient diagnosed with hyperekplexia. A missense mutation in the open reading frame of the GlyT2 gene inherited in homozygosity caused the substitution G449E in a residue highly conserved across the phylogenetic scale. The sequences of the glycine receptor genes GLRA1 and GLRB did not show abnormalities. We expressed the recombinant GlyT2 variant in heterologous cells and analyzed its pathogenic mechanism. The transporter was totally inactive, behaving as a bona fide loss-of-function mutant. Furthermore, the mutation promoted the abnormal insertion of the protein into the membrane, leading to its large incorporation into lipid rafts. However, there was no apparent alteration of wild-type trafficking upon mutant coexpression, as the mutant was prematurely degraded from the endoplasmic reticulum. Rescue with chemical chaperones was not possible for this mutant. Proteomics demonstrated that the expression of the mutant induced the unfolded protein response and interfered with raft-dependent processes. Therefore, the new variant causes a loss of function regarding GlyT2 activity but a gain of function as a cell proteostasis disturber. Full article

Parkinson’s disease (PD) is the second most prevalent neurodegenerative disease. It is characterized by mitochondrial dysfunction, increased reactive oxygen species (ROS), α-synuclein (α-syn) and phosphorylated-tau protein (p-tau) aggregation, and dopaminergic neuron cell death. Current drug therapies only provide temporary symptomatic relief and fail to stop or reverse disease progression due to the severe side effects or the blood–brain barrier. This study aimed to investigate the neuroprotective effects of an intermittent heating approach, thermal cycling-hyperthermia (TC-HT), in an in vitro PD model using rotenone (ROT)-induced human neural SH-SY5Y cells. Our results revealed that TC-HT pretreatment conferred neuroprotective effects in the ROT-induced in vitro PD model using human SH-SY5Y neuronal cells, including reducing ROT-induced mitochondrial apoptosis and ROS accumulation in SH-SY5Y cells. In addition, TC-HT also inhibited the expression of α-syn and p-tau through heat-activated pathways associated with sirtuin 1 (SIRT1) and heat-shock protein 70 (Hsp70), involved in protein chaperoning, and resulted in the phosphorylation of Akt and glycogen synthase kinase-3β (GSK-3β), which inhibit p-tau formation. These findings underscore the potential of TC-HT as an effective treatment for PD in vitro, supporting its further investigation in in vivo models with focused ultrasound (FUS) as a feasible heat-delivery approach. Full article

The 90 kDa heat shock proteins (Hsp90) are molecular chaperones that regulate the stability and maturation of numerous client proteins implicated in the regulation of cancer hallmarks. Despite the potential of pan-Hsp90 inhibitors as anticancer therapeutics, their clinical development has been hindered by on-target toxicities, particularly ocular and cardiotoxic effects, as well as the induction of pro-survival, compensatory heat shock responses. Together, these and other complications have prompted the development of isoform-selective Hsp90 inhibitors. In this review, we discuss the molecular bases for Hsp90 function and inhibition and emphasize recent advances in isoform-selective targeting. Importantly, we highlight how Hsp90 inhibition can sensitize tumors to cancer immunotherapy by enhancing antigen presentation, reducing immune checkpoint expression, remodeling the tumor microenvironment, and promoting innate immune activation. Special focus is given to Hsp90β-selective inhibitors, which modulate immunoregulatory pathways without eliciting the deleterious effects observed with pan-inhibition. Preclinical and early clinical data support the integration of Hsp90 inhibitors with immune checkpoint blockade and other immunotherapeutic modalities to overcome resistance mechanisms in immunologically cold tumors. Therefore, the continued development of isoform-selective Hsp90 inhibitors offers a promising avenue to potentiate cancer immunotherapy with improved efficacy. Full article

Heavy metals are among the most persistent and bioaccumulative pollutants in marine ecosystems, posing significant toxicological threats to fish via complex molecular and cellular disruptions. This review synthesizes current knowledge on the cascade of mechanistic responses in marine fish following HM exposure, which includes oxidative stress, modulation of antioxidant responses, activation of detoxification systems, DNA damage, inflammation, apoptosis, neuroendocrine disruption, and ultimately, cellular energy imbalance. In addition to established pathways, the review highlights recent advances in mechanistic understanding and biomarker development, including cellular stress responses, epigenetic regulation, metal homeostasis mechanisms, and novel molecular indicators. These mechanisms support the development of an integrated biomarker framework that combines classical indicators (e.g., antioxidant enzymes, metallothionein) with next-generation endpoints (e.g., miRNA profiles, gene-level responses of metal transporters or stress chaperones, epigenetic alterations). The interpretation of biomarker responses requires consideration of the exposure context, environmental variables, and physiological status to ensure accurate assessment of sublethal toxicity in field settings. By bridging mechanistic understanding with biomonitoring relevance, this review provides a comprehensive foundation for advancing molecular tools in pollution monitoring and risk assessment. Special emphasis is placed on biomarkers specific to heavy metal exposure, enhancing their diagnostic value relative to general stress indicators. Full article

Targeted degradation technologies, primarily referring to targeted protein degradation, have emerged as promising drug discovery strategies. In contrast to traditional “occupancy-driven” inhibition approaches, these technologies ingeniously leverage the cell’s endogenous degradation mechanisms to achieve specific elimination of disease-causing targets. Autophagy, a highly conserved cellular clearance pathway, possesses broad substrate recognition capabilities, enabling degradation of not only individual proteins but also protein aggregates, damaged organelles, and invading pathogens. Given these characteristics, researchers are actively exploring the application of autophagy mechanisms in targeted degradation technologies. Herein, we summarize recent advances in autophagy-dependent degradation approaches, including autophagosome tethering compounds (ATTEC), autophagy-targeting chimeras (AUTAC), autophagy-targeting Chimera (AUTOTAC), chaperone-mediated autophagy (CMA)-based methods, nanotechnology-based strategies, and the newly introduced autophagy-induced antibody (AUTAB) technique, highlighting their mechanisms, advantages, and potential applications in treating tumors, neurodegenerative diseases, and other challenging conditions. Full article

The Steinernema carpocapsae nematode is known to release several excretory/secretory products (ESPs) in its venom upon contact and during the parasitic infection process of insect hosts. A recurrent family of proteins found in this nematode’s venom is the CAP (cysteine-rich secretory protein/antigen 5/pathogenesis-related 1) protein, but the functional role of these proteins remains unknown. To elucidate the biological function, this study focused on characterising the secreted protein, first identified in the venom of the nematode’s parasitic stage, and the sequence retrieved from transcriptomic analysis. The structural comparisons of the Sc-CAP protein model, as determined by AlphaFold2, revealed related structures from other parasitic nematodes of vertebrates. Some of these closely related proteins are reported to have sterol-binding ability. The Sc-CAP recombinant protein was successfully produced in Escherichia coli in conjunction with a chaperone protein. The results showed that the Sc-CAP protein binds to cholesterol, and docking analyses of sterols on the protein revealed potential molecular interactions. Immunoassays performed in Galleria mellonella larvae revealed that this venom protein has an inhibitory effect against phenoloxidase and the antimicrobial response of insects. This suggests that the venom protein has an immunomodulatory function against insects, emphasising its importance during the parasite–host interaction. Full article