Abstract HSP90 is a highly conserved chaperone that facilitates the proliferation of many viruses, including silkworm ( bombyx mori ) nucleopolyhedrovirus (BmNPV), but the underlying regulatory mechanism was unclear. We found that suppression of HSP90 by 17‐AAG, a HSP90‐specific inhibitor, significantly reduced the expression of BmNPV capsid protein gp64 and viral genome replication, whereas overexpression of B. mori HSP90(BmHSP90) promoted BmNPV replication. Furthermore, in a recent study of the lysine acetylome of B. mori infected with BmNPV, we focused on the reduced viral proliferation due to changes of BmHSP90 lysine acetylation. Site‐directed introduction of acetylated (K/Q) or deacetylated (K/R) mimic mutations into BmHSP90 revealed that lysine 64 (K64) acetylation activated the JAK/STAT pathway and reduced BmHSP90 ATPase activity, leading to diminished chaperone activity and ultimately inhibiting BmNPV proliferation. In this study, a single lysine 64 acetylation change of BmHSP90 was elucidated as a model of posttranslational modifications occurring in the wake of host‐virus interactions, providing novel insights into potential antiviral strategies.
Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile.
ABSTRACT Alda‐1 functions as an agonist of aldehyde dehydrogenase (ALDH2) within the mitochondria, contributing to the preservation of mitochondrial structure and function. This study aimed to determine whether Alda‐1 inhibited the accumulation of mitochondrial reactive oxygen species (mtROS) and improved cardiomyocyte damage under oxidative stress. Cardiomyocytes derived from human induced pluripotent embryonic stem cells (iPSC‐CMs) and human AC16 cardiomyocytes were chosen for the experiments. Oxidative stress was induced in both cardiomyocyte types using hydrogen peroxide (H 2 O 2 ), a commonly employed agent. The mtROS accumulation and mitochondrial functional status were assessed by measuring the ROS content, mitochondrial membrane potential, and mitochondrial respiratory chain function. Co‐IP experiments were used to analyze whether mtROS promoted protein interactions between TXNIP and NLRP3 and induced NLRP3 inflammasome activation. The results showed that Alda‐1 mitigated damage to mitochondrial structure and function under oxidative stress, concurrently reducing the accumulation of mtROS. Co‐IP experiments revealed that elevated mtROS levels attenuated the protein interaction between TXNIP and TRX while intensifying the interaction between TXNIP and NLRP3. Consequently, this triggers activation of the NLRP3 inflammasome, leading to cardiomyocyte damage. In contrast, TXNIP knockdown inhibited H 2 O 2 ‐induced myocardial injury and enhanced the therapeutic effects of Alda‐1. Collectively, the results show that, in an H 2 O 2 environment, Alda‐1 increased the production of ALDH2 activity in cardiomyocytes, hindered the production of mtROS, disrupted the interaction between TXNIP and NLRP3, and alleviated myocardial damage during oxidative stress.
Storage protein 2 (SP2) not only is an important source of energy for the growth and development of silkworm but also has inhibitory effects on cell apoptosis. Endothelial cell (EC) apoptosis is an important contributing factor in the development of atherosclerosis; therefore, study of the antiapoptotic activity of SP2 on ECs provides information related to the treatment of atherosclerosis and other cardiovascular diseases. In this study, the sp2 gene was cloned and expressed in Escherichia coli to produce a 6xHis-tagged fusion protein, which was then used to generate a polyclonal antibody. Western blot results revealed that SP2 levels were higher in the pupal stage and hemolymph of fifth-instar larvae but low in the egg and adult stages. Subcellular localization results showed that SP2 is located mainly on the cell membrane. In addition, a Bac-to-Bac system was used to construct a recombinant baculovirus for SP2 expression. The purified SP2 was then added to a culture medium for human umbilical vein ECs (HUVECs), which were exposed to staurosporine. A cell viability assay demonstrated that SP2 could significantly enhance the viability of HUVEC. Furthermore, both ELISA and flow cytometry results indicated that SP2 has anti-apoptotic effects on staurosporine-induced HUVEC apoptosis.
Objective To explore the efficacy and safety of umbilical cord mesenchymal stem cells (UC-MSC) in the treatment of aplastic anemia (AA).Methods Eighteen patients with AA whose median age was 28 (7～49) were enrolled,including 10 severe AA; 7 of whom received immunosuppression treatment,but didn' t got good effect.UC-MSC were isolated from the umbilical cords of healthy fetuses and then cultured.The third to fifth generation cells were intravenously administered to the patients 1 × 106/kg once or twice a week.Before and after the infusion,complete blood cell counting,bone marrow aspiration,bone marrow biopsy,flow cytometry analysis of lymphocyte subsets,and clinical symptoms were observed.And treatmentrelated adverse reactions were observed.Results After a median of 38-time (12～96 times) infusions of UC-MSC 8-month(3-16 months) treatment,and 31-month (6～36 months) follow-up,3 cases were almost completely cured,7 cases relieved,1 case improved,2 cases got better in anemia and bleeding and longer transfusion interval,5 didn' t responded to the treatment,with a total response rate of 72.2％.4 of 7 frontline immunosuppressive-resistant patients responded to the treatment.7 of 12 patients with inverted CD4+/CD8+ recovered back to normal.No patient occurred adverse reaction.Conslusions UC-MSC in the treatment of AA is effective and safe and has no short term adverse reactions.It is a good therapy for the patients not responding to immunosuppression treatment,not suitable for transplant,or not able to tolerate the adverse reactions.
Key words:
Umbilical cord; Mesenchymal stem cells (MSC); Aplastic anemia (AA)
A key molecule in the pathogenesis of Alzheimer's disease (AD) is a 42-amino acid isoform of the amyloid-β peptide (Aβ42), which is the most toxic element of senile plaques. In this study, to develop an edible, safe, low-cost vaccine for AD, a cholera toxin B subunit (CTB)-Aβ42 fusion protein was successfully expressed in silkworm pupae. We tested the silkworm pupae-derived oral vaccination containing CTB-Aβ42 in a transgenic mouse model of AD. Anti-Aβ42 antibodies were induced in these mice, leading to a decreased Aβ deposition in the brain. We also found that the oral administration of the silk worm pupae vaccine improved the memory and cognition of mice, as assessed using a water maze test. These results suggest that the new edible CTB-Aβ42 silkworm pupae-derived vaccine has potential clinical application in the prevention of AD.
Objective To investigate the risk factors of ventriculo-peritoneal shunt tube blockage after surgery in infant with hydrocephalus and the effect of preoperative administration of umbilical cord mesenchymal stem cells(Mesenchymal stme cells,MSC) on the incidence of this complication.Methods A retrospective analysis was conducted in 125 cases of infant with hydrocephalus to evaluate the occurrence of shunt blockage during the follow-up of 2 years after ventriculo-peritoneal drainage.Further,the incidence and its associated factors in 11 cases who received intravenous infusion of MSC treatment preoperatively were also analyzed.Results The clinical risk factors included preoperative cerebrospinal fluid cell count,cerebrospinal fluid protein content,age,terminal of location drainage tube and serum albumin concentrations orderly.MSC treatment significantly increased serum albumin level and decreased the occurrence of drainage tube obstruction(1/11 vs.53/114,P0.01).Conclusion Preoperative infusion of MSC can increase the level of serum albumin and reduce post-operative complications.
4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA by attack of Asp145 on the C(4) of the substrate benzoyl ring to form a Meisenheimer intermediate (EMc), followed by expulsion of chloride ion to form an arylated enzyme intermediate (EAr) and, finally, ester hydrolysis in EAr to form 4-hydroxybenzoyl-CoA (4-HBA-CoA). This study examines the contribution of the active site His90 to catalysis of this reaction pathway. The His90 residue was replaced with glutamine by site-directed mutagenesis. X-ray crystallographic analysis of H90Q dehalogenase complexed with 4-HBA-CoA revealed that the positions of the catalytic groups are unchanged from those observed in the structure of the 4-HBA-CoA−wild-type dehalogenase complex. The one exception is the Gln90 side chain, which is rotated away from the position of the His90 side chain. The vacated His90 site is occupied by two water molecules. Kinetic techniques were used to evaluate ligand binding and catalytic turnover rates in the wild-type and H90Q mutant dehalogenases. The rate constants for 4-CBA-CoA (both 7 μM-1 s-1) and 4-HBA-CoA (33 and 11 μM-1 s-1) binding to the two dehalogenases are similar in value. For wild-type dehalogenase, the rate constant for a single turnover is 2.3 s-1 while that for multiple turnovers is 0.7 s-1. For H90Q dehalogenase, these rate constants are 1.6 × 10-2 and 2 × 10-4 s-1. The rate constants for EMc formation in wild-type and mutant dehalogenase are ∼200 s-1 while the rate constants for EAr formation are 40 and 0.3 s-1, respectively. The rate constant for hydrolysis of EAr in wild-type dehalogenase is 20 s-1 and in the H90Q mutant, 0.13 s-1. The 133-fold reduction in the rate of EAr formation in the mutant may be the result of active site hydration, while the 154-fold reduction in the rate EAr hydrolysis may be the result of lost general base catalysis. Substitution of the His90 with Gln also introduces a rate-limiting step which follows catalysis, and may involve renewing the catalytic site through a slow conformational change.
The ICAD gene plays an important role in the growth and development processesin insects. We conducted a molecular cloning and functional analysis to study a specific silkworm gene BmICAD related to apoptosis. The BmICAD gene was obtained from the fifth instar larvae of the silkworm by RT-PCR and over-expressed in Escherichia coli as His-tagged fusion proteins. Subcellular localization of the protein indicated that BmICAD was found in the cytoplasm near the nucleus. RNAi assay indicated that the apoptosis rate of Bm5 cells increased markedly. A His pull-down assay was used to investigate proteins that bind to rBmICAD. Two proteins, DNA supercoiling factor (SCF) and secreted protein acidic and rich in cysteine (SPARC), were found. These results indicate that down regulation of BmICAD can increase the apoptosis rate of Bm5 cells, and that SCF and SPARC may have important roles by interacting with BmICAD during this process. Key words: Bombyx mori, BmICAD, subcellular localization, RNAi, protein interaction.
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