Effect of Methyl-CpG Binding Domain Protein 2(MBD2) on AMD-like Lesions in ApoE-Deficient Mice

Summary： The role of methyl-CpG binding domain protein 2 （MBD2） in an ApoE-deficient mouse model of age-related macular degeneration （AMD） was investigated. Eight-week-old Mbd2/ApoE double deficient （Mbd2^-/- ApoE^-/-） mice （n=12, 24 eyes, experimental group） and MBD2 （wt） ApoE^-/- mice （n=12, 24 eyes, control group） were fed on Western-type diet for 4 months. The mice were sacrificed, and total serum cholesterol levels were analyzed and Bruch＇s membrane （BM） of the eyes was removed for ultrastructural observation by transmission electron microscopy. Moreover, intercellular adhesion molecule 1 （ICAM-1） immunoreactivities were evaluated by fluorescence microscopy in sections of the eyes in both groups for further understanding the function mechanism of MBD2. There was no significant difference in the total serum cholesterol levels between control group and experimental group （P〉0.05）. Transmission electron microscopy revealed that AMD-like lesions, various vacuoles accumulated on BM, notable outer collagenous layer deposits and dilated basal infoldings of retinal pigment epithelium （RPE） were seen in both groups, and the BM in control group was significantly thickened as compared with experimental group （P〈0.05）. Fluorescence micrographs exhibited the expression of ICAM-1 in choroid was higher in control group than in experimental group. We are led to conclude that MBD2 gene knockout may lead to accumulation of more deposits on the BM and influence the pathogenesis of AMD via triggering endothelial activation and inflammatory response in choroid, improving microcirculation, and reducing lipid deposition so as to inhibit the development of AMD-like lesions. Our study helps to provide a new therapeutic approach for the clinical treatment of AMD.

Mutual regulation between microRNA-373 and methyl-CpGbinding domain protein 2 in hilar cholangiocarcinoma

AIM:To investigate the reciprocal modulation between microRNA(miRNA) and DNA methylation via exploring the correlation between miR-373 and methyl-CpGbinding domain protein(MBD)2.METHODS:MiR-373 expression was examined using the TaqMan miRNA assay.Methylation of miR-373 was investigated using methylation-specific polymerase chain reaction,and recruitment of methyl binding proteins was studied using the chromatin immunoprecipitation assay.Mutation analysis was conducted using the QuikChange Site-Directed Mutagenesis kit.The activity of miR-373 gene promoter constructs and targeting at MBD2-three prime untranslated region(3'UTR) by miR-373 were evaluated by a dual-luciferase reporter gene assay.RESULTS:In hilar cholangiocarcinoma,miR-373 decreased and was closely associated with poor cell differentiation,advanced clinical stage,and shorter survival.The promoter-associated CpG island of miR-373 gene was hypermethylated and inhibited expression of miR-373.MBD2 was up-regulated and enriched at the promoter-associated CpG island of miR-373.Methylation-mediated suppression of miR-373 required MBD2 enrichment at the promoter-associated CpG island,and miR-373 negatively regulated MBD2 expression through targeting the 3'UTR.CONCLUSION:MiR-373 behaves as a direct transcriptional target and negative regulator of MBD2 activity through a feedback loop of CpG island methylation.

Methyl-CpG binding domain(MBD)2/3 specifically recognizes and binds to the genomic mCpG site with a β-sheet in the MBD to affect embryonic development in Bombyx mori

Methyl-CpG(mCpG)binding domain(MBD)proteins especially bind with methylated DNA,and are involved in many important biological processes;however,the binding mechanism between insect MBD2/3 and mCpG remains unclear.In this study,we identified 2 isoforms of the MBD2/3 gene in Bombyx mori,MBD2/3-S and MBD2/3-L.Binding analysis of MBD2/3-L,MBD2/3-S,and 7 mutant MBD2/3-L proteins deficient inβ1−β6 orα1 in the MBD showed thatβ2−β3-turns in theβ-sheet of the MBD are necessary for the formation of the MBD2/3–mCpG complex;furthermore,other secondary structures,namely,β4−β6 and anα-helix,play a role in stabilizing theβ-sheet structure to ensure that the MBD is able to bind mCpG.In addition,sequence alignment and binding analyses of different insect MBD2/3s indicated that insect MBD2/3s have an intact and conserved MBD that binds to the mCpG of target genes.Furthermore,MBD2/3 RNA interference results showed that MBD2/3-L plays a role in regulating B.mori embryonic development,similar to that of DNA methylation;however,MBD2/3-S withoutβ4−β6 andα-helix does not alter embryonic development.These results suggest that MBD2/3-L recognizes and binds to mCpG through the intactβ-sheet structure in its MBD,thus ensuring silkworm embryonic development.

A rapid nucleic acid detection platform based on phosphorothioate-DNA and sulfur binding domain

Nucleic acid detection plays a key role in diverse diagnosis and disease control.Currently available nucleic acid detection techniques are challenged by trade-offs among speed,simplicity,precision and cost.Here,we described a novel method,designated SENSOR(Sulfur DNA mediated nucleic acid sensing platform),for rapid nucleic acid detection.SENSOR was developed from phosphorothioate(PT)-DNA and sulfur binding domain(SBD)which specifically binds double-stranded PT-modified DNA.SENSOR utilizes PT-DNA oligo and SBD as targeting module,which is linked with split luciferase reporter to generate luminescence signal within 10 min.We tested detection on synthesized nucleic acid and COVID-19 pseudovirus,achieving attomolar sensitivity combined with an amplification procedure.Single nucleotide polymorphisms(SNP)could also be discriminated.Indicating SENSOR a new promising nucleic acid detection technique.

Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites

The outbreak of coronavirus disease(COVID-19)caused by SARS-CoV-2 virus continually lead to worldwide human infections and deaths.Currently,there is no specific viral protein-targeted therapeutics.Viral nucleocapsid protein is a potential antiviral drug target,serving multiple critical functions during the viral life cycle.However,the structural information of SARS-CoV-2 nucleocapsid protein remains unclear.Herein,we have determined the 2.7 A crystal structure of the N-terminal RNA binding domain of SARS-CoV-2 nucleocapsid protein.Although the overall structure is similar as other reported coronavirus nucleocapsid protein N-terminal domain,the surface electrostatic potential characteristics between them are distinct.Further comparison with mild virus type HCoV-OC43 equivalent domain demonstrates a unique potential RNA binding pocket alongside theβ-sheet core.Complemented by in vitro binding studies,our data provide several atomic resolution features of SARS-CoV-2 nucleocapsid protein N-terminal domain,guiding the design of novel antiviral agents specific targeting to SARS-CoV-2.

Identification of binding domains and key amino acids involved in the interaction between BmLARK and G4 structure in the BmPOUM2 promoter in Bombyx mori

It has been found that the non-B form DNA structures,like G-quadruplex(G4)and i-motif,are involved in many important biological processes.Our previous study showed that the silkworm transcription factor BmLARK binds to the G4 structure in the promoter of the transcription factor BmPOUM2 and regulates its promoter activity.However,the binding mechanism between BmLARK and BmPOUM2 G4 structure remains unclear.In this study,binding domains and key amino acid residues involved in the interaction between BmLARK and BmPOUM2 G4 were studied.The electrophoretic mobility shift assay results indicated that the two RNA-recognition motifs(RRM)of BmLARK are simultaneously required for the binding with the G4 structure.Either RRM1 or RRM2 alone could not bind with the G4 structure.The zinc-finger motif was not involved in the binding.A series of mutant proteins with specific amino acid mutations were expressed and used to identify the key amino acid residues involving the interaction.The results indicated thatβsheets,especially theβ1 andβ3 sheets,in the RRM domains of BmLARK played critical roles in the binding with the G4 structure.Several amino acid mutations of RRM1/2 in ribonucleoprotein domain 1(RNP1)(motif inβ3 strand)and RNP2(motif inβ1 strand)caused loss of binding ability,indicating that these amino acids are the key sites for the binding.All the results suggest that RRM domains,particularly their the RNP1 and RNP2 motifs,play important roles not only in RNA recognition,but also in the G4 structure binding.

Sequence difference of angiotensin-converting enzyme 2 between nonhuman primates affects its binding-affinity with SARS-CoV-2 S receptor binding domain

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)has caused many deaths and contributed to a tremendous public health concern worldwide since 2020.Angiotensin-converting enzyme 2(ACE2)binds to the SARS-CoV-2 virus as a receptor.The challenge of different nonhuman primate(NHP)species by SARSCoV-2 virus demonstrated different effects on virus replication and disease pathology.This study characterizes differences between host ACE2 sequences of three NHP species:Macaca mulatta,Macaca fascicularis,and Chlorocebus sabaeus.In addition,the binding affinity between the ACE2 ectodomain and the SARS-CoV-2 S receptor-binding domain(RBD)was analyzed.Variation of ACE2 sequence among NHP species and the binding affinity may account for different susceptibility and responses to SARS-CoV-2 infection.

Fragment antigen binding domains (F_(ab)s) as tools to study assembly-line polyketide synthases

The crystallization of proteins remains a bottleneck in our fundamental understanding of their functions.Therefore,discovering tools that aid crystallization is crucial.In this review,the versatility of fragment-antigen binding domains(F_(ab)s)as protein crystallization chaperones is discussed.F_(ab)s have aided the crystallization of membrane-bound and soluble proteins as well as RNA.The ability to bind three F_(ab)s onto a single protein target has demonstrated their potential for crystallization of challenging proteins.We describe a high-throughput workflow for identifying F_(ab)s to aid the crystallization of a protein of interest(POI)by leveraging phage display technologies and differential scanning fluorimetry(DSF).This workflow has proven to be especially effective in our structural studies of assembly-line polyketide synthases(PKSs),which harbor flexible domains and assume transient conformations.PKSs are of interest to us due to their ability to synthesize an unusually broad range of medicinally relevant compounds.Despite years of research studying these megasynthases,their overall topology has remained elusive.One F ab in particular,1B2,has successfully enabled X-ray crystallographic and single particle cryo-electron microscopic(cryoEM)analyses of multiple modules from distinct assembly-line PKSs.Its use has not only facilitated multidomain protein crystallization but has also enhanced particle quality via cryoEM,thereby enabling the visualization of intact PKS modules at near-atomic(3–5Å)resolution.The identification of PKS-binding F_(ab)s can be expected to continue playing a key role in furthering our knowledge of polyketide biosynthesis on assembly-line PKSs.

Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells

Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was～25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from～30 kD to～25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner.

Zinc finger E-box-binding homeobox 1 mediates aerobic glycolysis via suppression of sirtuin 3 in pancreatic cancer

AIM TO uncover the roles of tumor-promoting gene ZEB1 in aerobic glycolysis regulation and shed light on the underlying molecular mechanism.METHODS Endogenous zinc finger E-box binding homeobox-1 （ZEB1） was silenced using a and the impact of ZEB1 and lentivirus-mediated method, methyI-CpG binding domain protein 1 （MBD1） on aerobic glycolysis was measured using seahorse cellular flux analyzers, reactive oxygen species quantification, and mitochondrial membrane potential measurement. The interaction between ZEB1 and MBD1 was assessed by co-immunoprecipitation and immunofluorescence assays. The impact of ZEB1 and MBD1 interaction on sirtuin 3 （SIRT3） expression was confirmed by quantitative polymerase chain reaction, western blotting, and dual-luciferase and chromatinimmunoprecipitation assays.RESULTS ZEB1 was a positive regulator of aerobic glycolysis in pancreatic cancer. ZEB1 transcriptionally silenced expression of SIRT3, a mitochondrial-localized tumor suppressor, through interaction with MBD1.CONCLUSION ZEB1 silenced SIRT3 expression via interaction with MBD1 to promote aerobic glycolysis in pancreatic cancer.

Clinical and genetic characteristics of a child with Sotos syndrome and attention-deficit/hyperactivity disorder:A case report

BACKGROUND Sotos syndrome is an autosomal dominant disorder,whereas attention-deficit/hyperactivity disorder(ADHD)is a neurodevelopmental condition.This report aimed to summarize the clinical and genetic features of a pediatric case of Soros syndrome and ADHD in a child exhibiting precocious puberty.CASE SUMMARY The patient presented with accelerated growth and advanced skeletal maturation;however,she lacked any distinct facial characteristics related to specific genetic disorders.Genetic analyses revealed a paternally inherited heterozygous synonymous mutation[c.4605C>T(p.Arg1535Arg)].Functional analyses suggested that this mutation may disrupt splicing,and bioinformatics analyses predicted that this mutation was likely pathogenic.After an initial diagnosis of Sotos syndrome,the patient was diagnosed with ADHD during the follow-up period at the age of 8 years and 7 months.CONCLUSION The potential for comorbid ADHD in Sotos syndrome patients should be considered to avoid the risk of a missed diagnosis.

Molecular dynamics-driven exploration of peptides targeting SARS-CoV-2,with special attention on ACE2,S protein,M^(pro),and PL^(pro):A review

Molecular dynamics(MD)simulation is a computational technique that analyzes the movement of a system of particles over a given period.MD can provide detailed information about the fluctuations and conformational changes of biomolecules at the atomic level over time.In recent years,MD has been widely applied to the discovery of peptides and peptide-like molecules that may serve as severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)inhibitors.This review summarizes recent advances in such explorations,focusing on four protein targets:angiotensin-converting enzyme 2(ACE2),spike protein(S protein),main protease(M^(pro)),and papain-like protease(PL^(pro)).These four proteins are important druggable targets of SARS-CoV-2 because of their roles in viral entry,maturation,and infectivity of the virus.A review of the literature revealed that ACE2,S protein,and M^(pro) have received more attention in MD research than PL^(pro).Inhibitors of the four targets identified by MD simulations included peptides derived from food and other bioresources,peptides designed using the targets as templates,and peptide-like molecules retrieved from databases.Many of the inhibitors have yet to be validated in experimental assays for potency.Nevertheless,the role of MD simulation as an efficient tool in the early stages of anti-SARS-CoV-2 drug discovery agents has been demonstrated.

Prediction of Proteins Associated with COVID-19 Based Ligand Designing and Molecular Modeling

Current understanding about how the virus that causes COVID-19 spreads is largely based on what is known about similar coronaviruses.Some of the Natural products are suitable drugs against SARS-CoV-2 main protease.For recognizing a strong inhibitor,we have accomplished dock-ing studies on the major virus protease with 4 natural product species as anti COVID-19(SARS-CoV-2),namely“Vidarabine”,“Cytarabine”,“Gem-citabine”and“Matrine”which have been extracted fromGillan’s leaves plants.These are known as Chuchaq,Trshvash,Cote-Couto and Khlvash in Iran.Among these four studied compounds,Cytarabine appears as a suitable com-pound with high effectiveness inhibitors to this protease.Finally by this work we present a method on the Computational Prediction of Protein Structure Associated with COVID-19 Based Ligand Design and Molecular Modeling.By this investigation,auto dock software(iGEM-DOCK)has been used and via this tool,the suitable receptors can be distinguished in whole COVID-19 component structures for forming a complex.“iGEMDOCK”is suitable to define the binding site quickly.With docking simulation and NMR inves-tigation,we have demonstrated these compounds exhibit a suitable binding energy around 9 Kcal/mol with various ligand proteins modes in the bind-ing to COVID-19 viruses.However,these data need further evaluation for repurposing these drugs against COVID-19 viruses,in both vivo&vitro.

Therapeutic targeting of the androgen receptor(AR)and AR variants in prostate cancer

Prostate cancer(PCa)accounted for over 300000 deaths world-wide in 2018.Most of the PCa deaths occurred due to the aggressive castration-resistant PCa(CRPC).Since the androgen receptor(AR)and its ligands contribute to the continued growth of androgendependent PCa(ADPCa)and CRPC,AR has become a well-characterized and pivotal therapeutic-target.Although AR signaling was identified as therapeutic-target in PCa over five-decades ago,there remains several practical issues such as lack of antagonist-bound AR crystal structure,stabilization of the AR in the presence of agonists due to N-terminus and C-terminus interaction,unfavorable large-molecule accommodation of the ligand-binding domain(LBD),and generation of AR splice variants that lack the LBD that impede the discovery of highly potent fail-safe drugs.This review summarizes the AR-signaling pathway targeted therapeutics currently used in PCa and the approaches that could be used in future ARtargeted drug development of potent next-generation molecules.The review also outlines the discovery of molecules that bind to domains other than the LBD and those that inhibit both the full length and splice variant of ARs.

Effect of Antisense MBD1 Gene Eukaryotic Expression Plasmid on Expression of MBD1 Gene in Human Biliary Tract Carcinoma Cells

Hypermethylation of the promoter region is one of the major mechanism of tumor suppressor gene inactivation. In order to provide a research tool for the study on the function of MBD1 gene in DNA methylation and tumorigenesis, antisense MBD1 gene eukaryotic expression plasmid was constructed and transfected into human biliary tract carcinoma cell line QBC-939 to observe its effect on the expression of MBD1 mRNA and protein by using RT-PCR and FCM respectively. Following the transfection, the mRNA level of MBD1 gene decreased from 0. 912±0.022 to 0. 215±0. 017, and the protein level of MBD1 gene also decreased from （80.19±5.05） %to （35.11±4.05） %. There were very significant differences in the expression both at the transcription and post-transcription levels of MBD1 gene between non-tranfection group and the antisense MBD1 gene eukaryotic expression plasmid transfection group （P〈0.01）. It was suggested that transfection with the antisense MBD1 gene eukaryotic expression plasmid can significantly reduce the expression level of MBD1 gene in QBC-939, and this study may provide a valid tool for the investigation of the function of MBD1 gene and its role in biliary tract carcinoma.

Rapid characterization of the binding property of HtrA2/Omi PDZ domain by validation screening of PDZ ligand library

HtrA2/Omi is a mammalian mitochondrial serine protease, and was found to have dual roles in mam- malian cells, not only acting as an apoptosis-inducing protein but also maintaining mitochondrial ho- meostasis. PDZ domain is one of the most important protein-protein interaction modules and is in- volved in a variety of important cellular functions, such as signal transduction, degradation of proteins, and formation of cytoskeleton. Recently, it was reported that the PDZ domain of HtrA2/Omi might regulate proteolytic activity through its interactions with ligand proteins. In this study, we rapidly characterized the binding properties of HtrA2/Omi PDZ domain by validation screening of the PDZ ligand library with yeast two-hybrid approach. Then, we predicted its novel ligand proteins in human proteome and reconfirmed them in the yeast two-hybrid system. Finally, we analyzed the smallest networks bordered by the shortest path length between the protein pairs of novel interactions to evaluate the confidence of the identified interactions. The results revealed some novel binding proper- ties of HtrA2/Omi PDZ domain. Besides the reported Class II PDZ motif, it also binds to Class I and Class III motifs, and exhibits restricted variability at P?3, which means that the P?3 residue is selected according to the composition of the last three residues. Seven novel ligand proteins of HtrA2/Omi PDZ domain were discovered, providing significant clues for further clarifying the roles of HtrA2/Omi. Moreover, this study proves the high efficiency and practicability of the newly developed validation screening of candidate ligand library method for binding property characterization of peptide-binding domains.

Expression, purification and evaluation of N-terminal domain of AcAP5 with Factor Xa inhibitory activity

Ancylostoma anticoagulant peptide 5 （AcAP5） is a strong inhibitor of human coagulation factor Xa （FXa）. The N-terminal residues （N40） of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 （DE3）. Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside （IPTG）, and the interest protein was identified by SDS-PAGE and purified using one-step nickel （Ni） affinity chromatography. Under the optimal expres- sion condition （0.05 mM IPTG for 6 h at 37 ℃）, the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.

Isolation of cDNAs Enconding Two Distinct Transcription Factors Binding to DRE cis-acting Element Involved in Cold-and drought-induced Expression of Arabidopsis rd29A Gene

Previous study has defined DRE (dehydration responsive element) cis acting element and its important role in expressions of Arabidopsis rd29A gene under cold, dehydration and high salt stresses. In order to clarify the expression mechanism of rd29A gene, we isolated two cDNA clones that encoded DRE binding proteins ( DREB1 and DREB2 ) from cold and drought treated Arabidopsis plants, using DRE cis acting element in the promoter region of rd29A gene and yeast One Hybrid screening method. Experiments showed both DREB1 and DREB2 specifically interacted with DRE cis element. Homologous analysis showed no significant similarity between DREB1 and DREB2 in whole deduced amino acid sequences. However, both DREB1 and DREB2 proteins contained a conserved DNA binding domain (AP2/EREBP domain). Structural analysis of proteins also showed they had a nuclear localization signal (NLS) in their N terminal region and an acidic activation region in their C terminal region. AP2/EREBP domain is composed of 58 amino acids, which presents in a large family of plant genes encoding DNA binding proteins. We analyzed many plant transcription factors containing conserved AP2/EREBP domains. The 14th valine (V) and 19th glutamate (E) in the amino acid sequence of AP2/EREBP domains might be the consensus recognizing and binding to DRE cis element. Northern analysis indicated that DREB1 gene was induced by low temperature, whereas DREB2 gene was induced by dehydration and high salt stresses. Present studies suggest that the expression of rd29A gene under low temperature, dehydration and high salt stresses is regulated by DREB1 and DREB2 transcriptional factors in two separate signal transduction pathways, respectively.

Discovery of novel inhibitors of anti-apoptotic Bcl-2 proteins derived from Bim BH3 domain

The BH3 mimetics targeting the interaction between the BH3-only proteins and their prosurvival Bcl-2family proteins have shown enormous potential as cancer therapeutics. Herein, seven analogues targeting anti-apoptotic Bcl-2 proteins derived from the Bim BH3 domain via sequence simplification and/or modification are described. The in vitro binding affinity on anti-apoptotic Bcl-2 proteins and cell killing activity were evaluated. The results showed that analogues could significantly bind to target proteins and exhibited anti-cancer effect against three cancer cell lines. Of particular interest were the analogue SM-5（KD= 9.48 nmol/L for Bcl-2） and SM-6（KD= 0.08 nmol/L for Bcl-xL）, which exhibited improved binding affinity compared with the lead Bim（KD= 16.90 nmol/L for Bcl-2 and 22.2 nmol/L for Bcl-xL, respectively）. These results indicated that the peptide sequence containing the four hydrophobic side chains occupying pockets within the BH3-recognition cleft of anti-apoptotic Bcl-2 proteins might be the minimum sequence required for the bioactivity and the active core region of Bim. Promising inhibitors of anti-apoptotic Bcl-2 proteins with high bioactivity might be designed based on the active core.

The S190R mutation in the hemagglutinin protein of pandemic H1N1 2009 influenza virus increased its pathogenicity in mice

Human influenza viruses preferentially bind to sialic acid-α2,6-galactose (SAα2,6Gal) receptors, which are predominant in human upper respiratory epithelia, whereas avian influenza viruses preferentially bind to SAα2,3Gal receptors. However, variants with amino acid substitutions around the receptor-binding sites of the hemagglutinin (HA) protein can be selected after several passages of human influenza viruses from patients’ respiratory samples in the allantoic cavities of embryonated chicken eggs. In this study, we detected an egg-adapted HA S190R mutation in the pandemic H1N1 virus 2009 (pdmH1N1), and evaluated the effects of this mutation on receptor binding affinity and pathogenicity in mice. Our results revealed that residue 190 is located within the pocket structure of the receptor binding site. The single mutation to arginine at position 190 slightly increased the binding affinity of the virus to the avian receptor and decreased its binding to the long human α2,6-linked sialic acid receptor. Our study demonstrated that the S190R mutation resulted in earlier death and higher weight loss in mice compared with the wild-type virus. Higher viral titers at 1 dpi (days post infection) and diffuse damage at 4 dpi were observed in the lung tissues of mice infected with the mutant virus.