Prokaryotic expression and functional analysis of molecular chaperone Acr2 protein of Mycobacterium tuberculosis / 中国生物制品学杂志

@#Objective To express the molecular chaperone Acr2 protein of Mycobacterium tuberculosis(Mtb)in E.coli and analyze the function. Methods The recombinant plasmid pET-28a-Acr2 was transformed into competent E. coli BL21(DE3),and induced by IPTG. The expressed His-Acr2 protein was purified by Ni-NTA chromatography and SuperdexTM200 10/300 GL gel filtration chromatography to obtain Acr2 protein. The Acr2 protein was refolded by spontaneous refolding and reassembly after thermal denaturation(100 ℃ for 15 min)and chemical denaturation(8 mol/L urea,37 ℃ for 4 h).The secondary structure of Acr2 protein before and after denaturation-renaturation was detected by circular dichroism spectroscopy and non-denaturing SDS-PAGE,and the molecular chaperone function of Acr2 protein in vitro was detected by substrate binding assay. Results The purified Acr2 protein had the relative molecular mass of about 232 000,the purity of over 90%,and the concentration of about 2 mg/mL,which recovered its natural secondary structure after denaturationrenaturation,and formed stable complexes with the denatured malate dehydrogenase(MDH)at 48 ℃. Conclusion The Acr2protein can restore its natural molecular conformation with molecular chaperone activity in vitro after denaturation-renaturation treatment,providing a new strategy for the preparation of Mtb protein antigen with natural activity.

Migalastat como monoterapia oral para la enfermedad de Fabry

Contexto: la enfermedad de Fabry es una patología de depósito lisosomal poco frecuente, ligada al cromosoma X y causada por la deficiencia o ausencia de la enzima α-galactosidasa-A. La nefropatía, junto con la cardiopatía y el compromiso neurológico de la enfermedad, conduce a una muerte prematura. Objetivo: esta revisión describe la monoterapia oral con migalastat en pacientes con enfermedad de Fabry y mutaciones "amenables". Metodología: una chaperona farmacológica oral denominada migalastat (Galafold®), estabiliza y favorece el pasaje de formas mutadas "amenables" de la enzima hacia los lisosomas, aumentando así su actividad. Resultados: los estudios de fase III Facets y Attract demostraron seguridad y eficacia en comparación con las terapias de reemplazo enzimático disponibles, alcanzando estabilización de la función renal, reducción de la masa ventricular izquierda y estabilización del biomarcador plasmático Lyso-Gb3. Conclusiones: migalastat fue generalmente bien tolerado en ambos estudios. Publicaciones posteriores de extensión evidenciaron resultados similares, confirmando la seguridad y la eficacia, tanto en pacientes que previamente se encontraban con terapia de reemplazo enzimático y han sido rotados a migalastat, como también en pacientes que han iniciado migalastat como primer tratamiento.

Background: Fabry disease is a rare lysosomal storage disorder, linked to the X chromosome, and caused by the deficiency or absence of the enzyme α-galactosidase-A. Nephropathy together with heart disease and neurological involvement lead to premature death. Purpose: This review describes oral migalastat monotherapy in patients with Fabry disease and "amenable" mutations. Methodology: An oral pharmacological chaperone called Migalastat (Galafold®), stabilizes and facilitates the trafficking of "amenable" mutated forms of the enzyme to the lysosomes, thus increasing its activity. Results: The phase III FACETS and ATTRACT studies have demonstrated safety and efficacy compared to available enzyme replacement therapies; achieving renal function stabilization, reduction of left ventricular mass and maintenance of plasmatic Lyso-Gb3 levels. Conclusions: Migalastat was generally well tolerated in both trials. Subsequent extension publications showed similar results, confirming the safety and efficacy both in patients who were previously on enzyme replacement therapy and have been switched to migalastat, as well as in patients who have started migalastat as their first treatment.

Progress of relationship between autophagy and hepatocellular carcinoma / 肿瘤研究与临床

Autophagy is an intracellular degradation process that delivers cytoplasmic constituents to the lysosome. Abnormality of autophagy is related to many human diseases, which provides a new clue to the pathophysiology of human cancer. However, the role of autophagy in normal liver physiology and the pathogenesis of liver diseases need to be further clarified. This article reviews the role of autophagy in the occurrence and development of hepatocellular carcinoma and the molecular mechanisms.

Evolution and Functional Diversity of Microbial Cold Shock Proteins / 中国生物化学与分子生物学报

Cold shock proteins (CSPs) are highly conserved in structure and diversified in function. Due to the high homology of proteins, the CSP family mostly has a fixed spherical structure. CSPs are mostly the molecular chaperons of nucleic acid, so as to regulate the transcription and translation of genes, so that the cells can recover normal growth and reproduction under adverse environmental conditions. At present, many studies have shown that although CSPs share a high degree of homology, CSPs develop functional diversity to fight against a variety of stress after natural selection under different environmental conditions. For example, CSPC protein can inhibit the expression of related proteins in the type III secretory system, and CSPD protein is mainly suitable for regulating the growth and development of nutrient-deficient cells, while the functions of CSPH and CSPF proteins remain unclear. At first, this paper summarized the details of the high homology and the diversity between CSPs family members. Secondly, the phylogenetic tree for the genetic distance between different CSPs was analyzed. The functional diversity of the family and their mechanisms in transcription and translation levels were also summarized. Finally, the existing problems of the CSP family research and their application in agriculture, food and medical fields are reviewed.

Design of small molecules targeting molecular chaperone system: review and perspective / 药学学报

Molecular chaperone system, which mainly consist of heat shock proteins family and their cochaperones, is crucial for maintaining proteostasis in life. It assists in folding, maturation and ubiquitin-proteasome-mediated degradation of proteins, thus to play a key role in cell proliferation and apoptosis. Functional disorder of molecular chaperone system is highly relevant to occurrence and development of multiple diseases including cancers, autoimmune disease/inflammatory, infective diseases, neurodegenerative disease, etc. Therefore, molecular chaperone system has long been regarded as potential drug targets. In this review, we outline the progress in the design of small molecules targeting molecular chaperone system and analyze the features of small molecules with different mechanisms. Finally, we put forward expects about potential development directions for future drug design in this field.

Chaperone-mediated Autophagy Regulates Cell Growth by Targeting SMAD3 in Glioma / 神经科学通报·英文版

Previous studies suggest that the reduction of SMAD3 (mothers against decapentaplegic homolog 3) has a great impact on tumor development, but its exact pathological function remains unclear. In this study, we found that the protein level of SMAD3 was greatly reduced in human-grade IV glioblastoma tissues, in which LAMP2A (lysosome-associated membrane protein type 2A) was significantly up-regulated. LAMP2A is a key rate-limiting protein of chaperone-mediated autophagy (CMA), a lysosome pathway of protein degradation that is activated in glioma. We carefully analyzed the amino-acid sequence of SMAD3 and found that it contained a pentapeptide motif biochemically related to KFERQ, which has been proposed to be a targeting sequence for CMA. In vitro, we confirmed that SMAD3 was degraded in either serum-free or KFERQ motif deleted condition, which was regulated by LAMP2A and interacted with HSC70 (heat shock cognate 71 kDa protein). Using isolated lysosomes, amino-acid residues 75 and 128 of SMAD3 were found to be of importance for this process, which affected the CMA pathway in which SMAD3 was involved. Similarly, down-regulating SMAD3 or up-regulating LAMP2A in cultured glioma cells enhanced their proliferation and invasion. Taken together, these results suggest that excessive activation of CMA regulates glioma cell growth by promoting the degradation of SMAD3. Therefore, targeting the SMAD3-LAMP2A-mediated CMA-lysosome pathway may be a promising approach in anti-cancer therapy.

Effect of Modified Shengjiangsan on Gd-IgA1 in IgA Nephropathy Rats： Based on C1GALT1/C1GALT1C1 Signaling Pathway / 中国实验方剂学杂志

ObjectiveThe effect of modified Shengjiangsan on immunoglobulin A （IgA） nephropathy was observed. The microRNA-148b （miRNA-148b）， interleukin 6 （IL-6）， core 1 beta 1，3-galactosyltransferase （C1GALT1）， molecular chaperone Cosmc （core1β3-Gal-T-specific molecular chaperone C1GALT1C1）， and galactose-deficient IgA1 （Gd-IGA1） in serum and kidney tissues of IgA nephropathy rats were detected to explore the underlying mechanism. The result is expected to lay a scientific basis for clinical application of modified Shengjiangsan in the treatment of IgA nephropathy. MethodA total of 42 SPF male SD rats were randomized into the normal group （8rats） and modeling group （34 rats） with the random number table method. After one week of adaptive feeding， rats for modeling were given bovine serum albumin （BSA， gavage）， lipopolysaccharide （LPS， injection into tail vein）， carbon tetrachloride （CCl4， subcutaneous injection）， and castor oil to induce IgA nephropathy. After modeling， two rats were randomly selected to test the modeling outcome. Then the model rats were classified into the model group， low-dose Chinese medicine group （modified Shengjiangsan，6.27 g·kg-1）， high-dose Chinese medicine group （modified Shengjiangsan，12.54 g·kg-1）， and benazepril group （10 mg·kg-1） with the random number table method， 8 in each group. The administration （gavage， once a day） lasted 4 weeks. The 24-h urinary total protein （24 h-UTP） was detected at the end of the 1st， 9th， and 13th week of the experiment. At the 14th week， after anesthesia， femoral artery blood was collected and centrifugated. The supernatant was collected to detect albumin （ALB）， aspartate aminotransferase （AST）， alanine aminotransferase （ALT）， serum creatinine （SCr）， and blood urea nitrogen （BUN）. The expression levels of IL-6 and Gd-IGA1 were determined by enzyme-linked immunosorbent assay （ELISA）. Based on hematoxylin-eosin （HE）/Masson/periodic Schiff-methenamine silver （PASM） staining， the pathological changes of renal tissues were observed. Ultrastructural changes of glomeruli were observed by transmission electron microscopy. The expression of miRNA-148b， IL-6， C1GALT1， and C1GALT1C1 was detected by immunohistochemistry. The mesangial area of the glomeruli was observed by immunofluorescence. Real-time polymerase chain reaction （Real-time PCR） was employed to determine the mRNA levels of mirNA-148b， IL-6， C1GALT1， and C1GALT1C1， and Western blot was used to detect the protein levels of IL-6， C1GALT1， and C1GALT1C1. ResultCompared with normal group， the model group showed increase in the content of 24 h-UTP， SCr， ALT， IL-6， and GD-IGA1 （P<0.05）， decrease in ALB content （P<0.05）. Moreover， rats in the model group demonstrated hyperplasia of glomerular mesangial cells， thickening of mesangial area， podocyte foot process effacement， and a large number of granular IgA immune complex in the mesangial area. In addition， the model group showed increase in the expression of IL-6 in mesangial area and podocytes， decrease in the expression of C1GALT1 and C1GALT1C1 in mesangial area and podocytes， enhanced expression of IL-6 mRNA and miRNA-148b （P<0.01）， weakened expression of C1GALT1 mRNA and C1GALT1C1 mRNA （P<0.01）， rise of IL-6 protein expression （P<0.01）， and reduction in the protein expression of C1GALT1 and C1GALT1C1 （P<0.01）. Compared with the model group， modified Shengjiangsan decreased the content of 24 h-UTP， SCr， ALT， IL-6， and Gd-IGA1 （P<0.05） and increased the content of ALB （P<0.05， P<0.01）. Moreover， with the treatment of this Chinese medicine， the pathological damage was significantly alleviated and the deposition of IgA immune complex in basement membrane was reduced. The expression of IL-6 in the mesangial area and podocytes of rats was decreased， and the expression of C1GALT1 and C1GALT1C1 in the mesangial area and podocytes of rats was increased. Moreover， the expression of IL-6 mRNA and miRNA-148b was decreased （P<0.01）， and the expression of C1GALT1 mRNA and C1GALT1C1 mRNA was increased （P<0.01）. The protein expression of IL-6 was decreased （P<0.05， P<0.01）， and the protein expression of C1GALT1 and C1GALT1C1 was enhanced （P<0.05， P<0.01）. The Chinese medicine group showed obvious dose-effect trend. ConclusionModified Shengjiangsan may reduce the expression of miRNA-148b and IL-6 in serum and kidney tissue of IgA nephropathy rats， restore the expression of C1GALT1 and C1GALT1C1， and decrease the generation of Gd-IGA1， so as to reduce renal pathological damage and proteinuria， protect the kidney protection， and finally delay the disease progression. Moreover， the effect is enhanced with the rise of dose.

Research advances in new technologies in targeted protein degradation / 药学学报

In recent years, the targeted protein degradation technology has developed quickly, with proteolysis-targeting chimera (PROTAC) as the best-known strategy through exploring the ubiquitin-proteasome system. A number of new targeted protein degradation strategies have been emerging to expand the scope of protein degradation technology, including lysosome-targeting chimeras (LYTACs), autophagy-targeting chimeras (AUTACs), autophagosome-tethering compounds (ATTECs) and chimeras based on chaperone-mediated autophagy (CMA). The emerging methodologies have explored another important protein degradation system in eukaryotes-lysosomal systems, such as the endosome-lysosome pathway and the autophagy-lysosome pathway. This review summaries the mechanisms and features of different strategies for targeted protein degradation, with a special emphasis on the new targeted protein degradation technologies, such as their current status, advantages and limitations.

Perceptions and Willingness to Undertake Transvaginal Sonography by Women at the Sally Mugabe Central Hospital, Zimbabwe

ntroduction: The acceptability and willingness to undergo Transvaginal Sonography by the patients havegeneratedmixedreactionsindifferenthealthcare settings. There is little that is known aboutZimbabweanwomen'sperceptionsandwillingness to undergo Transvaginal Sonography and there are no specific guidelines to guide its use. Aim:To evaluate the perception and willingness to undergo Transvaginal Sonography among women attending Obstetrics and Gynaecology clinic at SallyMugabeCentralHospitalinHarare, Zimbabwe.Methods:Across-sectionalsurveyusingastructured questionnaire was conducted between 1 and 30 June 2022. Atotal of 170 women attending the obstetrics and gynaecology clinic at Sally MugabeCentralHospitalwereselectedbyconsecutive sampling to participate in the study.Results:The majority of women (81.76%) had no previous TVS experience, with an equally large proportion (60.84%) not having seen a TVS probe before. Most of the women would prefer female sonographers (85.37%) to conduct the examination. In addition, about 58% of the participants concurred that a chaperone should be present. Less than half of the females (47.93%) were eager to have a TVS done on them. Equally, just 45% of the females said they would encourage others to have a TVS scan. The difference in willingness to undergo a TVS study stratified by employment status was statistically significant (chi-square 7.26, p = 0.03). Conclusion: Our study findings revealed that a large proportion of females had no previous TVS experience, with an equally large proportion not having seen a TVS probe before. Only a sizeable proportion of women were willing to accept TVS provided it is conducted by female sonographers and or in the presence of a chaperone. The findings underscoretheimportanceofeducationandawareness of the benefits of TVS in terms of diagnosis on maternal health outcomes, in the Zimbabwean population.

Changes in molecular chaperone-mediated autophagy expression of developmental rats after recurrent-status seizures / 中华实用儿科临床杂志

Objective:To observe the expression of molecular chaperone-mediated autophagy in hippocampal neurons and its relationship with brain injury after recurrent-status seizures.Methods:Seven-day-old SD rats were divided into two groups according to simple randomization: the control group (NS group, 6 rats) and the recurrent-seizure group (RS group, 39 rats). Rats in the RS group were subjected to recurrent seizures after repeated inhalation of flurothyl, with 30 minutes once each day for consecutive 7 days.A total of 30 convulsive models were successfully established (9 rats that failed to establish models were discarded), and they were further divided into 0 h, 1.5 h, 3 h, 12 h and 24 h after the last seizure according to simple randomization, with 6 rats in each group.Western blot and reverse transcription-polymerase chain reaction (RT-PCR) were adopted for the observation of the expression of molecular chaperone-mediated autophagy markers [heat shock cognate protein 70 (Hsc70), lysosome-associated membrane protein type 2a (LAMP-2a), heat shock protein 40(HSP40) and heat shock protein 90(HSP90)] in hip-pocampal neurons, and apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL).Results:(1) RT-PCR and Western blot showed that, compared with the NS group, the expression of Hsc70, as a molecular chape-rone, started to increase at 1.5 h and continued until 24 h after the last seizure in the RS group ( P<0.05). HSP90 increased immediately after the last seizure and lasted until 24 h after the seizure ( P<0.01); the expression of HSP40 and LAMP-2a also showed high expression after the last seizure episode ( P<0.05). (2) The TUNEL method showed that the number of apoptotic cells in the hippocampal CA1 region increased significantly at 3 h (36.33±5.16)/40 field, 12 h (44.83±4.83)/40 field and 24 h (54.83±7.16)/40 field after the last seizure compared with NS group(15.16±2.48)/40 field ( P<0.01). (3) Pearson correlation analysis showed that the level of apoptosis in hippocampal CA1 region of rats after recurrent seizures was positively correlated with the expression of molecular chaperone marker molecules (Hsc70: r=0.734, P=0.001; LAMP2a: r=0.790, P<0.001). Conclusions:After recurrent seizures in developmental rats, the presence of increased expression of multiple molecular chaperone-mediated autophagy, which may positively correlate with apoptosis, may be involved in the process of brain injury.

Metformin activates chaperone-mediated autophagy and improves disease pathologies in an Alzheimer disease mouse model

Chaperone-mediated autophagy (CMA) is a lysosome-dependent selective degradation pathway implicated in the pathogenesis of cancer and neurodegenerative diseases. However, the mechanisms that regulate CMA are not fully understood. Here, using unbiased drug screening approaches, we discover Metformin, a drug that is commonly the first medication prescribed for type 2 diabetes, can induce CMA. We delineate the mechanism of CMA induction by Metformin to be via activation of TAK1-IKKα/β signaling that leads to phosphorylation of Ser85 of the key mediator of CMA, Hsc70, and its activation. Notably, we find that amyloid-beta precursor protein (APP) is a CMA substrate and that it binds to Hsc70 in an IKKα/β-dependent manner. The inhibition of CMA-mediated degradation of APP enhances its cytotoxicity. Importantly, we find that in the APP/PS1 mouse model of Alzheimer's disease (AD), activation of CMA by Hsc70 overexpression or Metformin potently reduces the accumulated brain Aβ plaque levels and reverses the molecular and behavioral AD phenotypes. Our study elucidates a novel mechanism of CMA regulation via Metformin-TAK1-IKKα/β-Hsc70 signaling and suggests Metformin as a new activator of CMA for diseases, such as AD, where such therapeutic intervention could be beneficial.

Advances in research progress on acid tolerance mechanism of Gram- negative bacteria mediated by molecular chaperone protein / 中国药科大学学报

@#The tolerance of microorganism under acid stress is of significant importance to the growth and industrial production of bacterial strain. When the bacterial cells are exposed to the external acid environment, proteins in periplasmic space are under higher acid stress and are thusmore susceptible to severe damage from acid than the intracellular protein, and these are also more susceptible to severe damage from acid than the intracellular protein. During the acid-resisting process of Gram-negative bacteria, in addition to the intracellular decarboxylase system, the molecular chaperone can also participate in the identification and protection of the space structure of protein as an important "correcting" mechanism. This paper, reviews the current researches on the function, structure, acid-resisting mechanism of variousmolecular chaperones, including HdeA, HdeB, DnaK and GroEL. Finally, the research progress of the acid-resisting mechanism among Gram-negative bacteria is summarized. Through in-depth investigation and analysis of the physiological adaptation strategy of molecular chaperone to the acid stress environment, this study can help to conduct physiological property modification of target strain and improve their viability and tolerance in acid stress environment, which has its important theoretical and practical significance.

Molecular chaperones facilitate soluble expression of recombinant non-toxic mutant CRM197 of diphtheria toxin in Escherichia coli / 生物工程学报

Diphtheria toxin is an ADP-ribosyltransferase toxic to human cells. Mutation of the active site in its catalytic domain eliminates the toxicity, but retains its immunogenicity. A non-toxic mutant of diphtheria toxin known as CRM197 protein has become an ideal carrier protein for conjugate vaccines. CRM197 can further improve its immunogenicity by cross-linking with other antigens, so it has good potential to find broad applications. Unfortunately, inclusion bodies are easily formed during the expression of recombinant CRM197 protein in Escherichia coli, which greatly reduces its yield. In order to address this problem, pG-KJE8 vector carrying molecular chaperones and plasmid pET28a-CRM197, were co-expressed in Escherichia coli. The results showed that the recombinant CRM197 protein was successfully expressed and appeared largely in inclusion bodies. The molecular chaperones DnaK, DnaJ, GrpE, GroES and GroEL5 expressed can facilitate correct and rapid folding of CRM197. Furthermore, it can also improve the recovery rate of soluble CRM197 protein. The soluble expression of CRM197 was maximized upon addition of 1.0 mmol/L IPTG, 0.5 mg L-arabinose, 5.0 ng/mL tetracycline and induction at 20oC for 16 h. The soluble CRM197 protein shows good immunoreactivity, demonstrating the molecular chaperones expressed from pG-KJE8 facilitated the soluble expression of CRM197 protein in E. coli.

Dynamic conformational flexibility and molecular interactions of intrinsically disordered proteins

In order to improve crop plants in terms of their yield, drought resistance, pest resistance, nutritional value, etc.,modern agriculture has relied upon plant genetic engineering. Since the advent of recombinant DNA technology, several tools have been used for genetic transformations in plants such as Agrobacterium tumefaciens,virus-mediated gene transfer, direct gene transfer systems such as electroporation, particle gun, microinjectionand chemical methods. All these traditional methods lack specificity and the transgenes are integrated atrandom sites in the plant DNA. Recently novel techniques for gene targeting have evolved such as engineerednucleases such as Zinc Finger Nucleases, Transcription Activator like effector nucleases, Clustered regularinterspaced short palindromic repeats. Other advances include improvement in tools for delivery of geneediting components which include carrier proteins, and carbon nanotubes. The present review focuses on thelatest techniques for target specific gene delivery in plants, their expression and future directions in plantbiotechnology

Effects of non-caloric ultrashort wave on the expression of CoQ10 and C1GALT1C1 in rats with cerebral ischemia reperfusion injury / 中南大学学报(医学版)

OBJECTIVES@#To examine the changes of coenzyme Q10 (CoQ10) and β-galactosyl transferase specific chaperone 1 (C1GALT1C1) in brain of rats with ischemic injury at different time points and to explore the protective mechanism of ultrashort wave (USW) on ischemic brain injury.@*METHODS@#Fifty SD rats were randomly divided into 5 groups (=10 per group): a sham group (control group) and 4 experimental group (ischemia for 2 h). The 4 experimental groups were set as a model 1 d group, a USW 1 d group, a model 3 d group and a USW 3 d group, respectively. Five rats were randomly selected for 2,3,5-triphenyltetrazoliumchloride (TTC) staining in each experimental group, and the remaining 5 rats were subjected to Western blotting and real-time PCR. The percentage of cerebral infarction volume and the relative expression level of CoQ10 and C1GALT1C1 in the brain were examined and compared.@*RESULTS@#The infarct volume percentage after TTC staining was zero in the sham group. With the progress of disease and USW therapy, the infarct volume percentage was decreased in the experimental groups (all <0.05); Western blotting and real-time PCR showed that the relative expression level of CoQ10 in the sham group was the highest, while in the experimental groups, the content of CoQ10 showed a upward trend with the extension of disease and USW therapy, with significant difference (all <0.05). The relative expression level of C1GALT1C1 in the sham group was the lowest, but in the experimental groups, they showed a downward trend with the extension of disease and USW therapy, with significant difference (all <0.05).@*CONCLUSIONS@#Non-caloric USW therapy may upregulate the expression of CoQ10 to suppress the expression of C1GALT1C1 in rats, leading to alleviating cerebral ischemic reperfusion injury.

The molecular mechanism of the effect of benzoαpyrene on autophagy of molecular chaperones under simulated hypoxia / 药学学报

In this study, the effect of benzo[α]pyrene (BaP) on chaperone-mediated autophagy (CMA) in a simulated hypoxia environment was observed and the relationship to heat shock protein 90 (HSP90) was clarified. With HSP90 inhibitor geldanamycin (GA) and HSP90α silenced, the mRNA and protein expression of hypoxia-inducible factor-1α (HIF-1α), HSP90, heat shock cognate protein 70 (HSC70), and lysosomal associated protein 2A (LAMP-2A) of A549 cells on hypoxic environment by BaP were tested. Alkaline comet experiment, immunofluorescence γ-H2AX focus experiment, quantitative real-time PCR (qPCR), and Western blot analyses were used to clarify the relationship between the DNA damage of different concentrations of BaP in A549 cells and the mRNA and protein expression of CMA-related factors. The results show that hypoxia can promote the expression of mRNA and protein of CMA-related factors in A549 cells. This study found that BaP has an inhibitory effect on CMA under the hypoxic environment. The inhibition or silencing of HSP90 will enhance the inhibitory effect of BaP on CMA. In a normoxic environment, BaP causes DNA damage and promotes CMA.

The sequential switch from macroautophagy to chaperone-mediated autophagy in Raji cells induced by starvation / 中国药理学通报

Aim To research the cross-talk and conversion between macroautophagy and chaperone-media-ted autophagy ( CMA) in cultured Burkitt lymphoma Raji cells induced by starvation. Methods The autophagic vacuoles were observed by fluorescence microscopy and confocal laser-scanning microscopy with monodansylcadaverine staining. The expression of autophagy associated-proteins were determined by West-em blot. Results Both macroautophagy and CMA were activated sequentially instead of simultaneously in starvation-induced Raji cells, and macroautophagy was quickly activated and peaked during the first hours of near baseline. With starvation persisted, CM A progressively increased along with the decline of macroautoph- A gy. Conclusions Macroautophagy and CMA are maximally activated during different stages of starvation. Activation of these two pathways is often sequential. The sequential switch from macroautophagy to CMA might be conducive to the adaption of cancer cells to miscellaneous intracellular or extracellular changes, maintaining their own growth and proliferation.

Proteomic analysis reveals dysregulated cell signaling in ejaculated spermatozoa from infertile men / 亚洲男科学杂志(英文版)

Dysfunctional sperm maturation is the primary reason for the poor sperm motility and morphology in infertile men. Spermatozoa from infertile men were fractioned on three-layer density gradient (80%, 60%, and 40%). Fraction 1 (F1) refers to the least mature stage having the lowest density, whereas the fraction 4 (F4) includes the most dense and morphologically mature motile spermatozoa. Fraction 2 (F2) and fraction 3 (F3) represent the intermediate stages. Proteins were extracted and separated by 1-dimensional gel. Bands were digested with trypsin and analyzed on a LTQ-Orbitrap Elite hybrid mass spectrometer system. Functional annotations of proteins were obtained using bioinformatics tools and pathway databases. A total of 1585 proteins were detected in the four fractions of spermatozoa. A dysregulated protein turnover and protein folding may lead to accumulation of defective proteins or proteins that otherwise would have been eliminated during the process of maturation, resulting in the impairment of sperm function. Aberrant chaperone expression may be a major contributing factor to the defective sperm function. Androgen receptor was predicted as a transcription regulator in one of the networks and the affected pathways were chaperone-mediated stress response, proteosomal pathway, and sperm function. The downregulation of key pathways and proteins which compromises the fertilizing potential of spermatozoa may provide insight into the mechanisms that lead to male infertility.

Effects of chaperone-mediated autophagy on the level of α-synuclein oligomers in Parkinson's disease cell model / 上海交通大学学报（医学版）

Objective • To investigate the effects of chaperone-mediated autophagy (CMA) on α-synuclein oligomers level in the Parkinson's disease (PD) cell model with impaired ubiquitin proteasome system (UPS). Methods • The PD cell model was established by adding the proteasome inhibitor lactacystin in the SK-N-SH cell line stably transfected with wild type α-synuclein. The levels of α-synuclein oligomers, lysosome-associated membrane protein type 2A (LAMP2A) and 70 kDa heat shock homologous protein (HSC70) were detected using Western blotting. CMA function was inhibited with LAMP2A siRNA, and its effects on α-synuclein oligomers and cell viability were detected. Furthermore, the interaction of LAMP2A with α-synuclein oligomers was detected by immunoprecipitation. Results • In the PD cell model, the levels of α-synuclein oligomers, and CMA related proteins, i.e. LAMP2A and HSC70, were increased. Inhibiting the expression of LAMP2A further increased α-synuclein oligomers level, while it decreased cell viability. Furthermore, LAMP2A could interact with α-synuclein oligomers. Conclusion • In the PD cell model, CMA is one of the pathways regulating α-synuclein oligomers level. Inhibiting CMA function can further increase the α-synuclein oligomers level and deteriorate cell survival.

Research progress of the relationship between heat shock protein 27 and related intestinal diseases / 解放军医学杂志

Heat shock proteins (HSP) are highly conserved proteins that protect cells and enhance the body's resistance to stress. Heat shock protein 27 (HSP27) also known as heat shock protein beta-1 (HSPB1) is a small molecular weight HSP that in humans is encoded by the HSPB1 gene. HSP27 exerts its effect mainly in the form of phosphorylation activity, and has a variety of important biological functions, such as chaperone activity, thermotolerance, anti-oxidation and inhibition of apoptosis. HSP27 was found to be highly expressed in a variety of intestinal diseases. This article reviews the structure, function, mechanism of action of HSP27 and the relationship between HSP27 and intestinal diseases, and explores the clinical value and application prospect of HSP27 in intestinal diseases.