Unveiling the dark side of glucose-regulated protein 78 (GRP78) in cancers and other human pathology: a systematic review.

Glucose-Regulated Protein 78 (GRP78) is a chaperone protein that is predominantly expressed in the lumen of the endoplasmic reticulum. GRP78 plays a crucial role in protein folding by assisting in the assembly of misfolded proteins. Under cellular stress conditions, GRP78 can translocate to the cell surface (csGRP78) were it interacts with different ligands to initiate various intracellular pathways. The expression of csGRP78 has been associated with tumor initiation and progression of multiple cancer types. This review provides a comprehensive analysis of the existing evidence on the roles of GRP78 in various types of cancer and other human pathology. Additionally, the review discusses the current understanding of the mechanisms underlying GRP78's involvement in tumorigenesis and cancer advancement. Furthermore, we highlight recent innovative approaches employed in downregulating GRP78 expression in cancers as a potential therapeutic target.

A Variant in TBCD Associated with Motoneuronopathy and Corpus Callosum Hypoplasia: A Case Report.

Mutations in the tubulin-specific chaperon D (TBCD) gene, involved in the assembly and disassembly of the α/ß-tubulin heterodimers, have been reported in early-onset progressive neurodevelopment regression, with epilepsy and mental retardation. We describe a rare homozygous variant in TBCD, namely c.881G>A/p.Arg294Gln, in a young woman with a phenotype dominated by distal motorneuronopathy and mild mental retardation, with neuroimaging evidence of corpus callosum hypoplasia. The peculiar phenotype is discussed in light of the molecular interpretation, enriching the literature data on tubulinopathies generated from TBCD mutations.

GroEL-Proteotyping of Bacterial Communities Using Tandem Mass Spectrometry.

Profiling bacterial populations in mixed communities is a common task in microbiology. Sequencing of 16S small subunit ribosomal-RNA (16S rRNA) gene amplicons is a widely accepted and functional approach but relies on amplification primers and cannot quantify isotope incorporation. Tandem mass spectrometry proteotyping is an effective alternative for taxonomically profiling microorganisms. We suggest that targeted proteotyping approaches can complement traditional population analyses. Therefore, we describe an approach to assess bacterial community compositions at the family level using the taxonomic marker protein GroEL, which is ubiquitously found in bacteria, except a few obligate intracellular species. We refer to our method as GroEL-proteotyping. GroEL-proteotyping is based on high-resolution tandem mass spectrometry of GroEL peptides and identification of GroEL-derived taxa via a Galaxy workflow and a subsequent Python-based analysis script. Its advantage is that it can be performed with a curated and extendable sample-independent database and that GroEL can be pre-separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to reduce sample complexity, improving GroEL identification while simultaneously decreasing the instrument time. GroEL-proteotyping was validated by employing it on a comprehensive raw dataset obtained through a metaproteome approach from synthetic microbial communities as well as real human gut samples. Our data show that GroEL-proteotyping enables fast and straightforward profiling of highly abundant taxa in bacterial communities at reasonable taxonomic resolution.

Novel Isoform DTX3c Associates with UBE2N-UBA1 and Cdc48/p97 as Part of the EphB4 Degradation Complex Regulated by the Autocrine IGF-II/IRA Signal in Malignant Mesothelioma.

EphB4 angiogenic kinase over-expression in Mesothelioma cells relies upon a degradation rescue signal provided by autocrine IGF-II activation of Insulin Receptor A. However, the identity of the molecular machinery involved in EphB4 rapid degradation upon IGF-II signal deprivation are unknown. Using targeted proteomics, protein-protein interaction methods, PCR cloning, and 3D modeling approaches, we identified a novel ubiquitin E3 ligase complex recruited by the EphB4 C tail upon autocrine IGF-II signal deprivation. We show this complex to contain a previously unknown N-Terminal isoform of Deltex3 E3-Ub ligase (referred as "DTX3c"), along with UBA1(E1) and UBE2N(E2) ubiquitin ligases and the ATPase/unfoldase Cdc48/p97. Upon autocrine IGF-II neutralization in cultured MSTO211H (a Malignant Mesothelioma cell line that is highly responsive to the EphB4 degradation rescue IGF-II signal), the inter-molecular interactions between these factors were enhanced and their association with the EphB4 C-tail increased consistently with the previously described EphB4 degradation pattern. The ATPase/unfoldase activity of Cdc48/p97 was required for EphB4 recruitment. As compared to the previously known isoforms DTX3a and DTX3b, a 3D modeling analysis of the DTX3c Nt domain showed a unique 3D folding supporting isoform-specific biological function(s). We shed light on the molecular machinery associated with autocrine IGF-II regulation of oncogenic EphB4 kinase expression in a previously characterized IGF-II+/EphB4+ Mesothelioma cell line. The study provides early evidence for DTX3 Ub-E3 ligase involvement beyond the Notch signaling pathway.

Plasma membrane-localized Hsp40/DNAJ chaperone protein facilitates OsSUVH7-OsBAG4-OsMYB106 transcriptional complex formation for OsHKT1;5 activation.

The salinization of irrigated land affects agricultural productivity. HIGH-AFFINITY POTASSIUM (K+ ) TRANSPORTER 1;5 (OsHKT1;5)-dependent sodium (Na+ ) transport is a key salt tolerance mechanism during rice growth and development. Using a previously generated high-throughput activation tagging-based T-DNA insertion mutant pool, we isolated a mutant exhibiting salt stress-sensitive phenotype, caused by a reduction in OsHKT1;5 transcripts. The salt stress-sensitive phenotype of this mutant results from the loss of function of OsDNAJ15, which encodes plasma membrane-localized heat shock protein 40 (Hsp40). osdnaj15 loss-of-function mutants show decreased plant height, increased leaf angle, and reduced grain number caused by shorter panicle length and fewer branches. On the other h'and, OsDNAJ15-overexpression plants showed salt stress-tolerant phenotypes. Intriguingly, salt stress facilitates the nuclear relocation of OsDNAJ15 so that it can interact with OsBAG4, and OsDNAJ15 and OsBAG4 synergistically facilitate the DNA-binding activity of OsMYB106 to positively regulate the expression of OsHKT1;5. Overall, our results reveal a novel function of plasma membrane-localized Hsp40 protein in modulating, alongside chaperon regulator OsBAG4, transcriptional regulation under salinity stress tolerance.

Reactive oxygen species accumulation is synchronised with growth inhibition of temperature-sensitive recAts polA Escherichia coli.

When combined with recombinase defects, chromosome breakage and double-strand break repair deficiencies render cells inviable. However, cells are viable when an SOS response occurs in recAts polA cells in Escherichia coli. Here, we aimed to elucidate the underlying mechanisms of this process. Transposon mutagenesis revealed that the hslO gene, a redox chaperone Hsp33 involved in reactive oxidative species (ROS) metabolism, was required for the suppression of recAts polA lethality at a restricted temperature. Recently, it has been reported that lethal treatments trigger ROS accumulation. We also found that recAts polA cells accumulated ROS at the restricted temperature. A catalase addition to the medium alleviates the temperature sensitivity of recAts polA cells and decreases ROS accumulation. These results suggest that the SOS response and hslO manage oxidative insult to an acceptable level in cells with oxidative damage and rescue cell growth. Overall, ROS might regulate several cellular processes.

Brain Neurons during Physiological Aging: Morphological Features, Autophagic and Mitochondrial Contribution.

Accumulating data suggest that the brain undergoes various changes during aging. Among them are loss of both white and gray matter, neurons and synapses degeneration, as well as oxidative, inflammatory, and biochemical changes. The above-mentioned age-related features are closely related to autophagy and mitochondria. Therefore, we aimed to reveal the most peculiar morphological features of brain nervous tissue and to characterize the expression of autophagy and mitochondrial immunohistochemical biomarkers in neurons of different human brain zones during aging. Counting the number of neurons as well as Microtubule-associated proteins 1A/1B light chain 3B (LC3B), Heat shock protein 70 (HSP70), Lysosome-associated membrane protein type 2A (LAMP2A), Alpha subunit of ATP synthase (ATP5A), and Parkinson disease protein 7 (DJ1) immunohistochemical staining were performed on FFPE samples of human prefrontal cortex, corpus striatum, and hippocampus obtained from autopsy. Statistical analysis revealed a loss of neurons in the studied elderly group in comparison to the young group. When the expression of macroautophagy (LC3B), chaperon-mediated autophagy (HSP70, LAMP2A), and mitochondrial respiratory chain complex V (ATP5A) markers for the young and elderly groups were compared, the latter was found to have a significantly higher rate of optical density, whilst there was no significance in DJ1 expression. These findings, while preliminary, suggest that both autophagy and mitochondria are involved in neuronal maintenance during aging and could indicate their potential role in adaptive mechanisms that occur in aging.

Mass-spectrometry analysis of the human pineal proteome during night and day and in autism.

The human pineal gland regulates day-night dynamics of multiple physiological processes, especially through the secretion of melatonin. Using mass-spectrometry-based proteomics and dedicated analysis tools, we identify proteins in the human pineal gland and analyze systematically their variation throughout the day and compare these changes in the pineal proteome between control specimens and donors diagnosed with autism. Results reveal diverse regulated clusters of proteins with, among others, catabolic carbohydrate process and cytoplasmic membrane-bounded vesicle-related proteins differing between day and night and/or control versus autism pineal glands. These data show novel and unexpected processes happening in the human pineal gland during the day/night rhythm as well as specific differences between autism donor pineal glands and those from controls.

Polymorphic SERPINA3-R124C reduces pathogenesis of its wild type by shortening the lifetime of oligomeric Aß.

Amyloid beta (Aß) 42 peptide accumulated in Alzheimer disease (AD) patients' brain, often colocalized with serine protease inhibitor family A member 3 (SERPINA3). Being a chaperon, SERPINA3 accelerated Aß42 fibrillization. While analyzing chaperon activity of human SERPINA3 polymorphisms, we found SERPINA3-R124C played a role in protecting cells from Aß42 cytotoxicity. SH-SY5Y cells exposed to Aß42 preincubated with wild-type SERPINA3 (SERPINA3-WT) resulted in extended toxicity leading cell death whereas Aß42 with SERPINA3-R124C resulted in less cytotoxicity. Transmission electron microscope and thioflavin T assay revealed that SERPINA3-R124C shortened lifetime of small soluble oligomer and maintained ß-sheet rich protofibril-like aggregates for longer time compared to that of with SERPINA3-WT. Western blot assay confirmed that SERPINA3-R124C converted Aß42 mostly into high molecular aggregates. Here, we demonstrate first time that polymorphic SERPINA3 acts as a benign chaperon by modulating the transition states of Aß42, which may contribute to the reduction of AD risk.

The molecular mechanisms of copper metabolism and its roles in human diseases.

Copper is an essential element in cells; it can act as either a recipient or a donor of electrons, participating in various reactions. However, an excess of copper ions in cells is detrimental as these copper ions can generate free radicals and increase oxidative stress. In multicellular organisms, copper metabolism involves uptake, distribution, sequestration, and excretion, at both the cellular and systemic levels. Mammalian enterocytes take in bioavailable copper ions from the diet in a Ctr1-dependent manner. After incorporation, cuprous ions are delivered to ATP7A, which pumps Cu+ from enterocytes into the blood. Copper ions arrive at the liver through the portal vein and are incorporated into hepatocytes by Ctr1. Then, Cu+ can be secreted into the bile or the blood via the Atox1/ATP7B/ceruloplasmin route. In the bloodstream, this micronutrient can reach peripheral tissues and is again incorporated by Ctr1. In peripheral tissue cells, cuprous ions are either sequestrated by molecules such as metallothioneins or targeted to utilization pathways by chaperons such as Atox1, Cox17, and CCS. Copper metabolism must be tightly controlled in order to achieve homeostasis and avoid disorders. A hereditary or acquired copper unbalance, including deficiency, overload, or misdistribution, may cause or aggravate certain diseases such as Menkes disease, Wilson disease, neurodegenerative diseases, anemia, metabolic syndrome, cardiovascular diseases, and cancer. A full understanding of copper metabolism and its roles in diseases underlies the identification of novel effective therapies for such diseases.

Origin and Evolution of the Human Bcl2-Associated Athanogene-1 (BAG-1).

Molecular chaperones, particularly the 70-kDa heat shock proteins (Hsp70s), are key orchestrators of the cellular stress response. To perform their critical functions, Hsp70s require the presence of specific co-chaperones, which include nucleotide exchange factors containing the BCL2-associated athanogene (BAG) domain. BAG-1 is one of these proteins that function in a wide range of cellular processes, including apoptosis, protein refolding, and degradation, as well as tumorigenesis. However, the origin of BAG-1 proteins and their evolution between and within species are mostly uncharacterized. This report investigated the macro- and micro-evolution of BAG-1 using orthologous sequences and single nucleotide polymorphisms (SNPs) to elucidate the evolution and understand how natural variation affects the cellular stress response. We first collected and analyzed several BAG-1 sequences across animals, plants, and fungi; mapped intron positions and phases; reconstructed phylogeny; and analyzed protein characteristics. These data indicated that BAG-1 originated before the animals, plants, and fungi split, yet most extant fungal species have lost BAG-1. Furthermore, although BAG-1's structure has remained relatively conserved, kingdom-specific conserved differences exist at sites of known function, suggesting functional specialization within each kingdom. We then analyzed SNPs from the 1000 genomes database to determine the evolutionary patterns within humans. These analyses revealed that the SNP density is unequally distributed within the BAG1 gene, and the ratio of non-synonymous/synonymous SNPs is significantly higher than 1 in the BAG domain region, which is an indication of positive selection. To further explore this notion, we performed several biochemical assays and found that only one out of five mutations tested altered the major co-chaperone properties of BAG-1. These data collectively suggest that although the co-chaperone functions of BAG-1 are highly conserved and can probably tolerate several radical mutations, BAG-1 might have acquired specialized and potentially unexplored functions during the evolutionary process.

Improvement of soluble expression of GM-CSF in the cytoplasm of Escherichia coli using chemical and molecular chaperones.

The most common approaches to improve soluble expression of heterologous proteins are applications of molecular chaperones such as DnaK, DnaJ, GrpE, GroEL and GroES. The aim of present study was to enhance soluble expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) in Escherichia coli by different approaches including modification of cultivation and induction conditions, and thermally, genetically and chemically enhancement of expression of cellular chaperones. To genetically enhance amount of molecular chaperones, co-expression of pET28-GM-CSF and pKJE7 plasmids was performed. The soluble expressed protein was affinity purified and subjected to endotoxin removal. Co-expression with molecular chaperones significantly increased soluble expression of GM-CSF. Addition of chemical chaperones and osmolytes like NaCl (0.5 M), sucrose (0.5 M), sorbitol (0.5 M) and MgCl2 (1 mM) to growing media could improve solubility of GM-CSF. Biological activity of purified GM-CSF was confirmed based on its proliferative effect on HL-60 cell lines. The approach developed in the present study can be applied to improve soluble expression of other recombinant protein proteins.

Synthesis and biological evaluation of thielocin B1 analogues as protein-protein interaction inhibitors of PAC3 homodimer.

The synthesis and biological evaluation of thielocin B1 analogues have been demonstrated. Fourteen analogues modified in the central core and terminal carboxylic acid moiety were concisely synthesized by simple esterification or etherification reaction. The evaluation of synthetic analogues as inhibitors of proteasome assembling chaperone (PAC) complexes (the PAC3 homodimer and PAC1/PAC2) revealed that the natural product-like bending structure and terminal carboxylic acid groups were crucial for its biological activity. Moreover, SAR and in silico docking studies indicated that all methyl groups on the diphenyl ether moiety of thielocin B1 contribute to the potent and selective inhibition of the PAC3 homodimer via hydrophobic interactions.

Therapeutic Opportunities of Targeting Histone Deacetylase Isoforms to Eradicate Cancer Stem Cells.

Cancer stem cells (CSCs), or tumor-initiating cells, are a small subset of cancer cells with the capacity for self-renewal and differentiation, which have been shown to drive tumor initiation, progression, and metastasis in many types of cancer. Moreover, therapeutic regimens, such as cisplatin and radiation were reported to induce the enrichment of CSCs, thereby conferring chemoresistance on cancer cells. Therefore, therapeutic targeting of CSCs represents a clinical challenge that needs to be addressed to improve patient outcome. In this context, the effectiveness of pan or class-I histone deacetylase (HDAC) inhibitors in suppressing the CSC population is especially noteworthy in light of the new paradigm of combination therapy. Evidence suggests that this anti-CSC activity is associated with the ability of HDAC inhibitors to target multiple signaling pathways at different molecular levels. Beyond chromatin remodeling via histone acetylation, HDAC inhibitors can also block key signaling pathways pertinent to CSC maintenance. Especially noteworthy is the ability of different HDAC isoforms to regulate the protein stability and/or activity of a series of epithelial-mesenchymal transition (EMT)-inducing transcription factors, including HIF-1α, Stat3, Notch1, ß-catenin, NF-κB, and c-Jun, each of which plays a critical role in regulating CSCs. From the translational perspective, these mechanistic links constitute a rationale to develop isoform-selective HDAC inhibitors as anti-CSC agents. Thus, this review aims to provide an overview on the roles of HDAC isoforms in maintaining CSC homeostasis via distinct signaling pathways independent of histone acetylation.

E46K Mutant α-Synuclein Is Degraded by Both Proteasome and Macroautophagy Pathway.

Genetic studies have revealed that rare mutations and multiplications of the gene locus in α-synuclein (α-syn) are implicated in the pathogenesis of Parkinson's disease (PD). However, the pathological effects of α-syn are still obscure. The neurotoxicity of α-syn is mainly determined by its protein levels, which depend on a balance between synthesis and degradation. Therefore, verifying the possible routes contributing to the clearance of α-syn is important for PD therapy. In this study, we established stable lines overexpressing human wild-type (WT) and E46K mutant α-syn in rat PC12 cells and investigated the degradation pathways of α-syn by using a panel of inhibitors and inducers of lysosome and proteasome function. We also monitored the degradation kinetics of α-syn by using cycloheximide to block protein synthesis. Our data showed that both proteasome and chaperon-mediated autophagy (CMA) are responsible for the degradation of the WT α-syn. Meanwhile, E46K mutant α-syn is mainly degraded by the proteasome and macroautophagy pathway. Compared with the WT protein, E46K mutant α-syn turned over more slowly in PC12 cells. In addition, overexpression of E46K mutant α-syn increased vulnerability of PC12 cells to apoptosis insults when compared with WT α-syn. Our findings may verify the possible routes contributing to the degradation of the E46K mutant α-syn.

Study of HSPB6: Insights into the Properties of the Multifunctional Protective Agent.

HSPB6(Heat shock protein B6), is also referred to as P20/HSP20. Unlike other many other members of sHSP(small Heat shock protein) family, which tend to form high-molecular-mass oligomers, in solution, human HSPB6 only forms dimers. However, it still exhibits chaperon-like activity comparable with that of HSPB5. It is expressed ubiquitously, with high and constitutive expression in muscular tissues. sHSPs characteristically function as molecular chaperones and HSPB6 also has a molecular chaperone activity. HSPB6 is up-regulated in response to diverse cellular stress or damage and protect cells from otherwise lethal conditions. HSPB6 is widely recognized as a principle mediator of cardioprotective signaling and recent studies have unraveled the protective role of HSPB6 in disease or injury to the central nervous system. Moreover, accumulating evidence has implicated HSPB6 as a key mediator of diverse vital physiological processes, such as smooth muscle relaxation, platelet aggregation. The versatility of HSPB6 can be explained by its direct involvement in regulating different client proteins and its ability to form heterooligomer with other sHSPs, which seems to be dependent on HSPB6 phosphorylation. This review focuses on the properties including expression and regulation pattern, phosphorylation, chaperon activity, multiple cellular targets of HSPB6, as well as its possible role in physical and pathological conditions.

Behind the curtain of tauopathy: a show of multiple players orchestrating tau toxicity.

tau, a microtubule-associated protein, directly binds with microtubules to dynamically regulate the organization of cellular cytoskeletons, and is especially abundant in neurons of the central nervous system. Under disease conditions such as Pick's disease, progressive supranuclear palsy, frontotemporal dementia, parkinsonism linked to chromosome 17 and Alzheimer's disease, tau proteins can self-assemble to paired helical filaments progressing to neurofibrillary tangles. In these diseases, collectively referred to as "tauopathies", alterations of diverse tau modifications including phosphorylation, metal ion binding, glycosylation, as well as structural changes of tau proteins have all been observed, indicating the complexity and variability of factors in the regulation of tau toxicity. Here, we review our current knowledge and hypotheses from relevant studies on tau toxicity, emphasizing the roles of phosphorylations, metal ions, folding and clearance control underlining tau etiology and their regulations. A summary of clinical efforts and associated findings of drug candidates under development is also presented. It is hoped that a more comprehensive understanding of tau regulation will provide us with a better blueprint of tau networking in neuronal cells and offer hints for the design of more efficient strategies to tackle tau-related diseases in the future.

Transcriptomic response of yeast cells to ATX1 deletion under different copper levels.

BACKGROUND: Iron and copper homeostatic pathways are tightly linked since copper is required as a cofactor for high affinity iron transport. Atx1p plays an important role in the intracellular copper transport as a copper chaperone transferring copper from the transporters to Ccc2p for its subsequent insertion into Fet3p, which is required for high affinity iron transport. RESULTS: In this study, genome-wide transcriptional landscape of ATX1 deletants grown in media either lacking copper or having excess copper was investigated. ATX1 deletants were allowed to recover full respiratory capacity in the presence of excess copper in growth environment. The present study revealed that iron ion homeostasis was not significantly affected by the absence of ATX1 either at the transcriptional or metabolic levels, suggesting other possible roles for Atx1p in addition to its function as a chaperone in copper-dependent iron absorption. The analysis of the transcriptomic response of atx1∆/atx1∆ and its integration with the genetic interaction network highlighted for the first time, the possible role of ATX1 in cell cycle regulation, likewise its mammalian counterpart ATOX1, which was reported to play an important role in the copper-stimulated proliferation of non-small lung cancer cells. CONCLUSIONS: The present finding revealed the dispensability of Atx1p for the transfer of copper ions to Ccc2p and highlighted its possible role in the cell cycle regulation. The results also showed the potential of Saccharomyces cerevisiae as a model organism in studying the capacity of ATOX1 as a therapeutic target for lung cancer therapy.

Theory of mass-independent fractionation of isotopes, phase space accessibility, and a role of isotopic symmetry.

Key experimental and theoretical features of mass-independent fractionation (MIF) of isotopes, also known as the η-effect, are summarized, including its difference from the exit channel zero-point energy difference effect. The latter exactly cancels in the MIF. One key experimental result is that the MIF for O3 formation is a low-pressure phenomenon and, moreover, that it decreases with increasing pressure of third bodies at pressures far below the "Lindemann fall-off" pressures for three-body recombination of O and O2. A possible origin of the MIF is discussed in terms of a role for isotopologue symmetry in intramolecular energy sharing. An explanation is suggested for the large difference in the fall-off pressure for recombination and the pressure for a large decrease in MIF, in terms of a difference between deactivating collisions and what we term here "symmetry-changing collisions". It is noted that the theory of the MIF involves four recombination rate constants and an equilibrium constant, for each trace isotope, seven rate constants in all and two equilibrium constants. A conceptual shortcut is noted. Experimental and computational information that may provide added insight into the MIF mechanism and tests is described.

MTOR-independent induction of autophagy in trabecular meshwork cells subjected to biaxial stretch.

The trabecular meshwork (TM) is part of a complex tissue that controls the exit of aqueous humor from the anterior chamber of the eye, and therefore helps maintaining intraocular pressure (IOP). Because of variations in IOP with changing pressure gradients and fluid movement, the TM and its contained cells undergo morphological deformations, resulting in distention and stretching. It is therefore essential for TM cells to continuously detect and respond to these mechanical forces and adapt their physiology to maintain proper cellular function and protect against mechanical injury. Here we demonstrate the activation of autophagy, a pro-survival pathway responsible for the degradation of long-lived proteins and organelles, in TM cells when subjected to biaxial static stretch (20% elongation), as well as in high-pressure perfused eyes (30mmHg). Morphological and biochemical markers for autophagy found in the stretched cells include elevated LC3-II levels, increased autophagic flux, and the presence of autophagic figures in electron micrographs. Furthermore, our results indicate that the stretch-induced autophagy in TM cells occurs in an MTOR- and BAG3-independent manner. We hypothesize that activation of autophagy is part of the physiological response that allows TM cells to cope and adapt to mechanical forces.