Deciphering Functional Significance of Substrate-Binding Domain of Ssa1 on Heat-Shock Response and Prion Propagation

The Hsp70 (70kDa heat shock protein) is highly conserved in all species and has been implicated in a variety of important cellular functions such as heat shock response, prion propagation, protein folding and refolding, translocation across membranes and assembly of macromolecular complexes. A variety of evidence has accumulated to show that Hsp70 machinery is a key modulator of the stress response, such as heat shock and oxidant stress. Moreover, many human neurodegenerative diseases such as Alzheimer’s, Parkinson’s and the prion disease Creutzfeldt-Jacob Disease (CJD) are also intimately linked to Hsp70. Structurally, Hsp70 is comprised of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD). In this work, a well-established yeast system and a combination of computational biology, genetics, structural biology, biochemistry and molecular biology were utilized to decipher the role of the SBD of Hsp70 in regulation of heat shock response and [PSI+] prion propagation. It was found that mutations (F475S and L483W) located in a region termed β6-β7 dramatically decreased the stability of the SBD and the size of side chain contributes to maintain the hydrophobic core of SBD. Introduction of smaller amino acid side chains at residue 475, such as alanine and serine, resulted in temperature sensitivity and [PSI+] impairment. When the side chain of residue 475 is larger than Cysteine, no matter if it is polar or nonpolar (tyrosine and phenylalanine), thermotolerance and [PSI+] can be maintained. However, there is a limit to side-chain size as too large, such as tryptophan, Ssa1 will lose intrinsic function and fail to support cell viability. By contrast, residue 483 prefer to smaller size amino acid to remain the hydrophobic core of SBD. Therefore, mutations on those two residues easily disturb the integrity of SBD and thus promote the degradation of SBD in vitro and in vivo. Inter-domain communication between the NBD and SBD is affected through disruption of the important hydrophobic core and disturbance of a critical interface between the two domains. Disruption of the SBD structure abolishes repression of ATP hydrolysis of the NBD, reduces protein refolding activity and alters interactions with co-chaperones, especially Hsp104 and Hsp26, but decreased the interactions with Sup35. Degradation of the SBD in vivo is dependent on the action of vacuolar carboxypeptidase (Pep4) rather than the proteasome and occurs in WT cells at high temperature. Finally, SBD degradation is negatively regulated by the acetylation of four reversible hyperacetylated lysine residues, K86, K185, K354 and K562 of Ssa1. And, the thermotolerance regulation is independent from [PSI+] prion and alteration of the genome-wide translation caused by the read-through. The reversible hyperacetylated residues did not influence the basal expression level of the Hsp70 machinery, but rapidly respond the heat-shock stress by deacetylating themselves to stabilize SBD of Hsp70

Deciphering Functional Significance of Substrate-Binding Domain of Ssa1 on Heat-Shock Response and Prion Propagation

The Hsp70 (70kDa heat shock protein) is highly conserved in all species and has been implicated in a variety of important cellular functions such as heat shock response, prion propagation, protein folding and refolding, translocation across membranes and assembly of macromolecular complexes. A variety of evidence has accumulated to show that Hsp70 machinery is a key modulator of the stress response, such as heat shock and oxidant stress. Moreover, many human neurodegenerative diseases such as Alzheimer’s, Parkinson’s and the prion disease Creutzfeldt-Jacob Disease (CJD) are also intimately linked to Hsp70. Structurally, Hsp70 is comprised of two domains: nucleotide-binding domain (NBD) and substrate-binding domain (SBD). In this work, a well-established yeast system and a combination of computational biology, genetics, structural biology, biochemistry and molecular biology were utilized to decipher the role of the SBD of Hsp70 in regulation of heat shock response and [PSI+] prion propagation. It was found that mutations (F475S and L483W) located in a region termed β6-β7 dramatically decreased the stability of the SBD and the size of side chain contributes to maintain the hydrophobic core of SBD. Introduction of smaller amino acid side chains at residue 475, such as alanine and serine, resulted in temperature sensitivity and [PSI+] impairment. When the side chain of residue 475 is larger than Cysteine, no matter if it is polar or nonpolar (tyrosine and phenylalanine), thermotolerance and [PSI+] can be maintained. However, there is a limit to side-chain size as too large, such as tryptophan, Ssa1 will lose intrinsic function and fail to support cell viability. By contrast, residue 483 prefer to smaller size amino acid to remain the hydrophobic core of SBD. Therefore, mutations on those two residues easily disturb the integrity of SBD and thus promote the degradation of SBD in vitro and in vivo. Inter-domain communication between the NBD and SBD is affected through disruption of the important hydrophobic core and disturbance of a critical interface between the two domains. Disruption of the SBD structure abolishes repression of ATP hydrolysis of the NBD, reduces protein refolding activity and alters interactions with co-chaperones, especially Hsp104 and Hsp26, but decreased the interactions with Sup35. Degradation of the SBD in vivo is dependent on the action of vacuolar carboxypeptidase (Pep4) rather than the proteasome and occurs in WT cells at high temperature. Finally, SBD degradation is negatively regulated by the acetylation of four reversible hyperacetylated lysine residues, K86, K185, K354 and K562 of Ssa1. And, the thermotolerance regulation is independent from [PSI+] prion and alteration of the genome-wide translation caused by the read-through. The reversible hyperacetylated residues did not influence the basal expression level of the Hsp70 machinery, but rapidly respond the heat-shock stress by deacetylating themselves to stabilize SBD of Hsp70

Safety of azithromycin in paediatrics: a systematic review protocol

Introduction: Azithromycin is widely used in children not only in the treatment of individual children with infectious diseases, but also as mass drug administration (MDA) within a community to eradicate or control specific tropical diseases. MDA has also been reported to have a beneficial effect on child mortality and morbidity. However, concerns have been raised about the safety of azithromycin, especially in young children. The aim of this review is to systematically identify the safety of azithromycin in children of all ages.Methods and analysis: MEDLINE, PubMed, Cochrane Central Register of Controlled Trials, Embase, CINAHL, International Pharmaceutical Abstracts and adverse drug reaction (ADR) monitoring systems will be systematically searched for randomised controlled trials (RCTs), cohort studies, case–control studies, cross-sectional studies, case series and case reports evaluating the safety of azithromycin in children. The Cochrane risk of bias tool, Newcastle-Ottawa and quality assessment tools, and The Joanna Briggs Institute Critical Appraisal tools will be used for quality assessment. Meta-analyses will be conducted to the incidence of ADRs from RCTs if appropriate. Subgroup analyses will be performed in different age and azithromycin dosage groups

Above-knee Prosthesis Control Based on Posture Recognition by Support Vector Machine

Abstract-In order for individuals suffering from transfemoral amputation to walk in a variety of circumstances, the above-knee prosthesis based on posture recognition was presented. The body posture of lower limb was classified into four classes, "stair", "sitting", "standing", and "walking". For measure the amputee's movement intent, surface EMG signals which can reflect amputee's movement intent and can be measured without invasion were applied to identify postural adjustments by support vector machine. The result of this study indicates that this method can recognize every postural adjustment with a higher identification rate, and has a great potential in practical application of artificial lower limb

Ligand-binding properties of XaffOBP9, a Minus-C odorant-binding protein from Xyleborus affinis (Coleoptera: Curculionidae: Scolytinae)

Xyleborus affinis, one of the most important pests of rubber trees, has caused severe damage to the natural rubber industry in Hainan province. The ability to detect host plants through a sensitive and specific olfactory system is crucial for Xyleborus affinis. Odorant binding proteins (OBPs) are believed to bind and carry hydrophobic active compounds from the environment to the surface of olfactory receptor neurons. To investigate the potential functional role of the highly expressed XaffOBP9 in binding with semiochemicals, we cloned and analyzed the cDNA sequence of XaffOBP9. The results showed that XaffOBP9 contains a 411bp open reading frame that encodes 136 amino acids. Then XaffOBP9 was expressed in Escherichia coli. The binding affinity of the recombinant OBP to 15 different ligands (14 host plant volatiles and 1 aggregation pheromone) was then examined using a fluorescence competitive binding approach. The results demonstrated that XaffOBP9 exhibited broad binding capabilities and strong affinities for 14 ligands. The structure of XaffOBP9 and its interactions with fourteen ligands were further analyzed by modeling and molecular docking, respectively. Based on the docking result, we found hydrophobic interactions are important between XaffOBP9 to these ligands and three amino acid residues (L71, Y106, and L114) were highly overlapped and contributed to the interaction with ligands. Mutation functional assays confirmed that the mutant L114A showed significantly reduced binding capacity to these ligands. This study suggested that XaffOBP9 may be involved in the chemoreception of semiochemicals and that it is helpful for the integrated management of X. affinis

Protectin conjugates in tissue regeneration 1 alleviates sepsis-induced acute lung injury by inhibiting ferroptosis

Background: Acute lung injury (ALI) is a common and serious complication of sepsis with high mortality. Ferroptosis, categorized as programmed cell death, contributes to the development of lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is an endogenous lipid mediator that exerts protective effects against multiorgan injury. However, the role of PCTR1 in the ferroptosis of sepsis-related ALI remains unknown. Methods: A pulmonary epithelial cell line and a mouse model of ALI stimulated with lipopolysaccharide (LPS) were established in vitro and in vivo. Ferroptosis biomarkers, including ferrous (Fe2+), glutathione (GSH), malondialdehyde (MDA) and 4-Hydroxynonenal (4-HNE), were assessed by relevant assay kits. Glutathione peroxidase 4 (GPX4) and prostaglandin-endoperoxide synthase 2 (PTGS2) protein levels were determined by western blotting. Lipid peroxides were examined by fluorescence microscopy and flow cytometry. Cell viability was determined by a CCK-8 assay kit. The ultrastructure of mitochondria was observed with transmission electron microscopy. Morphology and inflammatory cytokine levels predicted the severity of lung injury. Afterward, related inhibitors were used to explore the potential mechanism by which PCTR1 regulates ferroptosis. Results: PCTR1 treatment protected mice from LPS-induced lung injury, which was consistent with the effect of the ferroptosis inhibitor ferrostatin-1. PCTR1 treatment decreased Fe2+, PTGS2 and lipid reactive oxygen species (ROS) contents, increased GSH and GPX4 levels and ameliorated mitochondrial ultrastructural injury. Administration of LPS or the ferroptosis agonist RSL3 resulted in reduced cell viability, which was rescued by PCTR1. Mechanistically, inhibition of the PCTR1 receptor lipoxin A4 (ALX), protein kinase A (PKA) and transcription factor cAMP-response element binding protein (CREB) partly decreased PCTR1 upregulated GPX4 expression and a CREB inhibitor blocked the effects ofPCTR1 on ferroptosis inhibition and lung protection. Conclusion: This study suggests that PCTR1 suppresses LPS-induced ferroptosis via the ALX/PKA/CREB signaling pathway, which may offer promising therapeutic prospects in sepsis-related ALI

Inhibition the ubiquitination of ENaC and Na,K-ATPase with erythropoietin promotes alveolar fluid clearance in sepsis-induced acute respiratory distress syndrome

Sepsis-induced acute respiratory distress syndrome (ARDS) causes significant fatalities worldwide and lacks pharmacological intervention. Alveolar fluid clearance (AFC) plays a pivotal role in the remission of ARDS and is markedly impaired in the pathogenesis of ARDS. Here, we demonstrated that erythropoietin could effectively ameliorate lung injury manifestations and lethality, restore lung function and promote AFC in a rat model of lipopolysaccharide (LPS)-induced ARDS. Moreover, it was proven that EPO-induced restoration of AFC occurs through triggering the total protein expression of ENaC and Na,K-ATPase channels, enhancing their protein abundance in the membrane, and suppressing their ubiquitination for degeneration. Mechanistically, the data indicated the possible involvement of EPOR/JAK2/STAT3/SGK1/Nedd4–2 signaling in this process, and the pharmacological inhibition of the pathway markedly eliminated the stimulating effects of EPO on ENaC and Na,K-ATPase, and subsequently reversed the augmentation of AFC by EPO. Consistently, in vitro studies of alveolar epithelial cells paralleled with that EPO upregulated the expression of ENaC and Na,K-ATPase, and patch-clamp studies further demonstrated that EPO substantially strengthened sodium ion currents. Collectively, EPO could effectively promote AFC by improving ENaC and Na,K-ATPase protein expression and abundance in the membrane, dependent on inhibition of ENaC and Na,K-ATPase ubiquitination, and resulting in diminishing LPS-associated lung injuries