Accurate and efficient protein sequence design through learning concise local environment of residues.

MOTIVATION: Computational protein sequence design has been widely applied in rational protein engineering and increasing the design accuracy and efficiency is highly desired. RESULTS: Here, we present ProDESIGN-LE, an accurate and efficient approach to protein sequence design. ProDESIGN-LE adopts a concise but informative representation of the residue's local environment and trains a transformer to learn the correlation between local environment of residues and their amino acid types. For a target backbone structure, ProDESIGN-LE uses the transformer to assign an appropriate residue type for each position based on its local environment within this structure, eventually acquiring a designed sequence with all residues fitting well with their local environments. We applied ProDESIGN-LE to design sequences for 68 naturally occurring and 129 hallucinated proteins within 20 s per protein on average. The designed proteins have their predicted structures perfectly resembling the target structures with a state-of-the-art average TM-score exceeding 0.80. We further experimentally validated ProDESIGN-LE by designing five sequences for an enzyme, chloramphenicol O-acetyltransferase type III (CAT III), and recombinantly expressing the proteins in Escherichia coli. Of these proteins, three exhibited excellent solubility, and one yielded monomeric species with circular dichroism spectra consistent with the natural CAT III protein. AVAILABILITY AND IMPLEMENTATION: The source code of ProDESIGN-LE is available at https://github.com/bigict/ProDESIGN-LE.

The unique Akt inhibitor SC66 suppressed AMPK activity and abolished autophagy through the EGFR-p62 pathway.

Akt is usually considered to be a negative regulator of both autophagy and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling. In the present study, we found that SC66, a pyridine-based allosteric Akt inhibitor, suppressed basal and H2 O2 -induced autophagy concurrent with decreased phosphorylation and activity of AMPK. SC66 treatment led to the formation of a high molecular weight (HMW) form of SQSTM1/p62 (p62), which is an autophagic substrate and is essential for selective autophagy. Moreover, we observed that SC66 inhibited the binding of p62 and microtubule-associated protein light chain 3 (LC3). The immunoprecipitation results revealed the interaction between p62 and epidermal growth factor receptor (EGFR), and knockdown of EGFR reversed SC66-mediated autophagy inhibition without affecting the phosphorylation of acetyl-CoA carboxylase (ACC), a well-known substrate of AMPK. SC66 increased the interaction between EGFR and Beclin 1 and markedly decreased the association of EGFR with VPS34, a critical protein for autophagy induction. Collectively, the data presented here indicate that EGFR-p62 pathway plays a critical role in Akt-mediated positive regulation of autophagy.

Preoperative CBCT Guided Bone Regeneration Combined with Concentrated Growth Factor Applied in the Implantation of Maxillary Teeth with Insufficient Bone Mass.

It was to explore the application effect of cone beam computed tomography (CBCT) guided bone regeneration (GBR) combined with concentrated growth factor (CGF) in the implantation of maxillary teeth with insufficient bone mass. 78 patients with single maxillary anterior tooth loss and labial alveolar defects in Dazhong Stomatological Hospital were retrospectively analyzed and randomly divided into groups A and B. Both groups were treated with surgical methods of GBR at the same time of implantation. Group A: CGF fibrin fluid and Bio-Oss bone powder, covered with CGF membrane on the surface to guide bone regeneration. Group B: Bio-Oss bone powder was implanted and covered with Heal All collagen biofilm. The horizontal bone width, keratinized gingival width, bone plate thickness, implant margin results, and alveolar ridge width were analyzed at 3 and 6 months before and after the operation in both groups. 6 months after the operation, the difference in the indexes between the two groups was evident; and in group A, the indexes had an obvious difference in contrast with those before operation; the healing rate of soft tissue was 100% in group A and 80% in group B, which was significantly different (P < 0.05). Preoperative CBCT GBR combined with CGF can promote the bone formation content of guided regeneration of bone defects in the maxillary teeth region.

Chloropupukeananin and Pestalofone C Regulate Autophagy through AMPK and Glycolytic Pathway.

Chloropupukeananin (RN56-6) and Pestalofone C (RN56-49), isolated from the culture of the plant endophytic fungus Pestalotiopsis fici, have been shown cytotoxic, anti-HIV, and antimicrobial activities. However, the underlying mechanism of their regulatory roles in autophagy remains unknown. In the present study, we revealed that both compounds increased the formation of autophagosome and enhanced autophagic flux. While RN56-6 upregulated the expression of HK2, one of the key rate-limiting enzymes of glycolysis, the inhibition of glycolysis chemically attenuated RN56-6-induced autophagy. On the contrary, RN56-49 downregulated the expression of HK2, while the suppression of glycolysis promoted RN56-49-dependent autophagic flux. Moreover, the knockdown of AMPKß1, a scaffolding subunit of AMPK, decreased autophagy induced by these two compounds. Collectively, these findings revealed that RN56-6 and RN56-49 regulated autophagic process through AMPK and glycolytic pathway.

[PFKFB3 regulates 11'-deoxyverticillin A (C42)-induced autophagy and apoptosis in HeLa cells].

OBJECTIVE: The aim is to reveal the role of PFKFB3 in 11'-deoxyverticillin A (C42)-induced autophagy and apoptosis. METHODS: Electron and fluorescence microscopy, immunoblotting, MTS assay, siRNA interference and real time PCR were used. RESULTS: C42 could induce multiple cell death in HeLa cells. Knockdown of either Beclin 1 or LC3, two important autophagic genes, increased both PARP-1 cleavage and cell viability loss. Although high dose of C42 triggered more cell viability loss, yet, it failed to augment autophagic flux. While PFKFB3 inhibitors attenuated C42-induced autophagy, the overexpression of PFKFB3 increased the induced autophagic flux. CONCLUSION: PFKFB3 is involved in C42 induced-autophagy, which blunts the caspase-dependent apoptotic process.

De novo generation of infectious prions with bacterially expressed recombinant prion protein.

The prion hypothesis is strongly supported by the fact that prion infectivity and the pathogenic conformer of prion protein (PrP) are simultaneously propagated in vitro by the serial protein misfolding cyclic amplification (sPMCA). However, due to sPMCA's enormous amplification power, whether an infectious prion can be formed de novo with bacterially expressed recombinant PrP (rPrP) remains to be satisfactorily resolved. To address this question, we performed unseeded sPMCA with rPrP in a laboratory that has never been exposed to any native prions. Two types of proteinase K (PK)-resistant and self-perpetuating recombinant PrP conformers (rPrP-res) with PK-resistant cores of 17 or 14 kDa were generated. A bioassay revealed that rPrP-res(17kDa) was highly infectious, causing prion disease in wild-type mice with an average survival time of about 172 d. In contrast, rPrP-res(14kDa) completely failed to induce any disease. Our findings reveal that sPMCA is sufficient to initiate various self-perpetuating PK-resistant rPrP conformers, but not all of them possess in vivo infectivity. Moreover, generating an infectious prion in a prion-free environment establishes that an infectious prion can be formed de novo with bacterially expressed rPrP.

The disulphide cleavage derivative (C42-4) of 11'-deoxyverticillin A (C42) fails to induce apoptosis and genomic instability in HeLa cells.

Our previous study revealed 11'-deoxyverticillin A (C42), a natural product isolated from the Ophiocordyceps-associated fungus Clonostachys rogersoniana and a member of the epipolythiodioxopiperazines (ETPs), induced both apoptosis and autophagy in HCT116 cells; however, the role of disulphide/polysulphide bridges of C42 in the regulation of autophagy remains unexplored. Here, we revealed that C42 activated both caspase-dependent apoptosis and autophagy in HeLa cells, whereas its disulphide cleavage derivative C42-4 failed to induce the cleavage of both caspase-3 and PARP-1. In contrast, both C42 and C42-4 increased the formation of autophagosomes, punctate staining of LC3, and the ratio of LC3-II to actin, suggesting that disulphide/polysulphide bridges are dispensable for the induction of the autophagic process. Moreover, we found that C42 but not C42-4 led to nuclear instability by increasing the formation of micronuclei and expression of phosphorylated histone H2AX (γ-H2AX), a widely used marker for DNA double strand breaks (DSBs), while Rad51, a protein pivotal for DNA repair, was decreased upon challenge with C42. These results demonstrate that the disulphide bonds in ETPs play an essential role in the induction of caspase-dependent apoptosis and nuclear stability.

[Effect of autophagy on expression of interferon in hepatitis B virus-infected hepatocytes].

OBJECT: Autophagy is a lysosomal degradation pathway in which eukaryotic cells dispose intracellular aggregates or defective organelles to maintain cellular homeostasis. Autophagy not only plays a key role in the growth, development, mature and differentiation of cells, but also is associated with pathogenesis, virus infection and immunity. To clarify the mechanism of Hepatitis B virus (HBV) infection and cell immune response, we investigated the relationship between autophagy and IFN factors in the HBV infected cells. METHODS: We inhibited the autophagy by the RNA interference knockdown of Beclin1 and Atg7, the essential autophagic genes, examined the number of autophagosomes by fluorescence microscopy and examined the expression of interferon factors by Real-Time PCR. RESULTS: Autophagy was inhibited after transfected siBeclin1 or siAtg7. After inhibiting the autophagy, the expression of interferon factors were decreased, but cell apoptosis was not induced. CONCLUSION: When the autophagy was inhibited, interferon signaling pathways were impaired in the HBV infected cells. The finding indicated that HBV induced-autophagy enhanced the interferon signaling pathways, and then increased the native immune response.

The Studies in Constructing Yeast Cell Factories for the Production of Fatty Acid Alkyl Esters.

Fatty acid alkyl esters have broad applications in biofuels, lubricant formulas, paints, coatings, and cosmetics. Traditionally, these esters are mostly produced through unsustainable and energy-intensive processes. In contrast, microbial production of esters from renewable and sustainable feedstocks may provide a promising alternative and has attracted widespread attention in recent years. At present, yeasts are used as ideal hosts for producing such esters, due to their availability for high-density fermentation, resistance to phage infection, and tolerance against toxic inhibitors. Here, we summarize recent development on the biosynthesis of alkyl esters, including fatty acid ethyl esters (FAEEs), fatty acid short-branched chain alkyl esters (FASBEs), and wax esters (WEs) by various yeast cell factories. We focus mainly on the enzyme engineering strategies of critical wax ester synthases, and the pathway engineering strategies employed for the biosynthesis of various ester products. The bottlenecks that limit productivity and their potential solutions are also discussed in this review.

[Infection with hepatitis B virus enhances basal autophagy].

OBJECTIVE: Hepatitis B virus (HBV) is a major human pathogen that chronically infects 400 million people worldwide. Chronic infection of HBV plays a key role in the pathogenesis of cirrhosis and hepatocellular carcinoma (HCC). To clarify the mechanism of HBV-related HCC and immune escape of HBV, we investigated the relationship between infection of HBV and basal autophagy. METHODS: To examine the number of autophagosomes upon transfection with HBV, HepG2.2.15 cells were transfected with green fluorescent protein-microtubule-associated protein 3 (GFP-LC3) and observed by fluorescence microscopy. Huh7 or HepG2 cells was transiently transfected with HBV expression vector pHBV1.3, then the phosphatidylethanol-amine conjugation of microtubule-associated protein 3 (LC3) and the degradation of p62, both of which are specific indictors of autophagy,were evaluated by western blot. Moreover, HepG2 or Chang liver cells were transiently transfected with the constructed HBV X protein ( HBx) expression vector in order to evaluate autophagic status of the cells. RESULTS: HBV and HBx were both able to increase the autophagosomes formation as well as the enhancement of autophagic flux. Notably, C type HBx had a more increment of autophagy than B type does. CONCLUSION: HBV can enhance basal autophagy and the increment is dependent on HBx. Different genotypes of HBx had different effects on basal autophagy. Take together, these findings will help us to clarify the mechanism of HBV infection and the development of Hepatitis B to HCC in the future study.

Distinct role of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication.

AIMS: To investigate the role and underlying mechanism of 4E-BP1 and S6K1 in regulating autophagy and hepatitis B virus (HBV) replication. MAIN METHODS: The mRNA relative expression of HBx and its DNA level were detected by real-time PCR. The relative levels of hepatitis B surface antigen (HBsAg) were measured by enzyme-linked immunosorbent assay (ELISA). HBx DNA level of HepG2 cells transfected with pcDNA3.1(+)-HBV1.3 plasmids were detected by Southern blot. Moreover, we determined autophagy through electron microscopy, confocal microscopy and Western blot. KEY FINDINGS: Rapamycin promoted autophagy and the X protein synthesis concomitantly with elevation in Akt phosphorylation and Beclin1 expression. Either Beclin1 or Akt depletion suppresses the Rapa-enhanced HBV replication, whereas mTOR silencing inhibited HBV replication concurring with a decreased in both S6K1 and 4E-BP1 phosphorylation. Unexpectedly, Akt inhibitor suppressed Rapa-dependent autophagic flux and increased the level of p62/SQSTM1. While S6K1 ablation impaired autophagy and decreased X protein expression, 4E-BP1 silencing slightly influenced autophagy and increased X protein level. SIGNIFICANCE: The underlying mechanism of 4E-BP1 and S6K1, two main downstream effectors of mTOR, in mediating HBV replication and HBV-induced autophagy remains largely unknown. Here, we propose that Akt is required for both HBV replication and Rapa-induced autophagy, and 4E-BP1 and S6K1 play a distinct role in the virus replication and autophagic process.

SQSTM1/p62 loss reverses the inhibitory effect of sunitinib on autophagy independent of AMPK signaling.

Sunitinib (ST), a multitargeted receptor tyrosine kinase inhibitor, has been demonstrated to be effective for the treatment of renal carcinoma. It has been reported that ST is involved in the mediation of autophagy; however, its regulatory role in the autophagic process remains controversial. Furthermore, the mechanism by which activated AMP-activated protein kinase (AMPK) negatively regulates autophagy remains nearly unexplored. In the present study, we revealed that ST inhibited AMPK activity and regulated autophagy in a cell type- and dose-dependent manner. In a number of cell lines, ST was demonstrated to inhibit H2O2-induced autophagy and the phosphorylation of acetyl-CoA carboxylase (ACC), whereas alone it could block the autophagic flux concurrent with increased expression of p62. An immunoprecipitation assay revealed that LC3 directly interacted with p62, whereas ST increased punctate LC3 staining, which was well colocalized with p62. Taken together, we reveal a previously unnoticed pathway for ST to regulate the autophagic process, and p62, although often utilized as a substrate in autophagy, plays a critical role in regulating the inhibition of ST in both basal and induced autophagy.

Grb2 binds to PTEN and regulates its nuclear translocation to maintain the genomic stability in DNA damage response.

Growth factor receptor bound protein 2 (Grb2) is an adaptor protein critical for signal transduction and endocytosis, but its role in DNA damage response (DDR) remains unknown. Here, we report that either knockdown of Grb2 or overexpression of the mutated Grb2 promotes micronuclei formation in response to oxidative stress. Furthermore, Grb2 was demonstrated to interact with phosphatase and tensin homologue (PTEN; a tumor suppressor essential for nuclear stability), and the loss of Grb2 reduced the nuclear-localized PTEN, which was further decreased upon stimulation with hydrogen peroxide (H2O2). Overexpression of the T398A-mutated, nuclear-localized PTEN reduced micronuclei frequency in the cells deficient of functional Grb2 via rescuing the H2O2-dependent expression of Rad51, a protein essential for the homologous recombination (HR) repair process. Moreover, depletion of Grb2 markedly decreased the expression of Rad51 and its interaction with PTEN. Notably, Rad51 showed a preference to immunoprecipation with the T398A-PTEN mutant, and silencing of Rad51 alone accumulated micronuclei concurring with decreased expression of both Grb2 and PTEN. Our findings indicate that Grb2 interacts with PTEN and Rad51 to regulate genomic stability in DDR by mediating the nuclear translocation of PTEN to affect the expression of Rad51.

Analysis of a point mutation in H5N1 avian influenza virus hemagglutinin in relation to virus entry into live mammalian cells.

Binding to and infection of human cells is essential for avian influenza virus transmission. Since virus binding is not always predictive for efficient infection of the cells, here we wished to investigate how hemagglutinin (HA) mutations of avian influenza virus H5N1 influence virus post-binding events in a single cycle of replication. One mutation observed in H5 HA of avian and natural human isolates from mainland China, Hong Kong, Vietnam and Thailand was identified and analyzed. The effects of the mutation on receptor binding, fusion and virus entry into cultured cells were investigated using hemadsorption, polykaryon formation and pseudotyped virus that express luciferase in the cytoplasm of transduced cell. Our results revealed that replacing aspartic acid at residue 94 with asparagine enhanced virus fusion activity and increased the binding of HA to sialic acid alpha2,6 galactose, while it decreased pseudotyped virus entry into cells expressing the avian type receptor, sialic acid alpha2,3 galactose. Our result may have implications for the understanding of the role of HA mutations in virus entry into live cells that exclusively display one type of receptor.

A dual reporter gene based system to quantitate the cell fusion of avian influenza virus H5N1.

Membrane fusion is central to the entry of influenza virus into host cells. To quantitatively determine the fusion activity of hemagglutinin (HA) of avian influenza virus H5N1, we established a cell fusion assay based on a dual luciferase reporter gene. The HA fusion activity was assayed by measuring luciferase expression in fused cells, allowing a rapid, sensitive, and quantitative comparison of HA fusion activities at various pHs and in different cells types. The simplicity and the quantitative nature of this novel assay are ideally suited for identifying viral receptors or screening for inhibitors of viral entry in the future.

In vitro self-propagation of recombinant PrPSc-like conformation generated in the yeast cytoplasm.

Self-propagation is characteristic property for a prion conformation. Previous studies revealed that prion protein expressed in the cytoplasm gained a PrP(Sc)-like conformation. However, it remains unclear whether the PrP(Sc)-like conformation has the self-propagating property. We found that PrP partially purified from yeast cytoplasm formed amyloid fiber like structures, and we found that the PrP(Sc)-like conformation is able to convert normal PrP(C) in the brain homogenate to a proteinase K-resistant conformation. These results suggest that yeast cytoplasm expressed recombinant PrP(Sc)-like conformation has the characteristic self-propagating property of a prion, which may have implications in the pathogenesis of sporadic and inherited prion diseases.

[Progress of miRNA and its functions in eukaryotes].

Two major kinds of non-coding RNAs play important roles in eukaryotes. One is microRNA (miRNA), the other is small interference RNA (siRNA). miRNA, 19-25 nt in length, functions in regulation of gene expression and development. Many kinds of miRNAs and some proteins assemble in a complex, miRNP, where miRNA gives its function, siRNA directs the target mRNA in RNA interference (RNAi). Some distinct differences are existed between miRNA and siRNA. The regulation mechanism of miRNA may be conserved in eukaryotes.

[11'-Deoxyverticillin A induces caspase-dependent cell apoptosis in PC3M cells].

Recent years, the incidence and mortality of prostate cancer have increased dramatically in China. At earlier stages, most diagnosed prostate cancers are responsive to androgen depletion treatment, yet, nearly all patients will eventually progress to metastatic androgen-independent prostate cancer (AIPC), which still has no effective therapeutic method or drug to deal with. 11'-Deoxyverticillin A (C42) belongs to the family of epipolythiodioxopiperazines (ETPs), an interesting class of fungal toxins that inhibit farnesyl transferase. Compounds holding such a property have been explored as putative anticancer agents. In this study, using PC3M cells, an AIPC cell line, we investigated the effect of the compound on apoptosis and explored the underlying mechanism. It revealed that C42 markedly enhanced the activity of caspase-3/7 and increased the accumulation of the cleaved PARP, all of which are the markers of apoptosis. It also revealed that C42 either decreased cell viability or inhibited the growth of PC3M cells. Moreover, we observed that the loss of cell viability and cell growth inhibition induced by C42 were both time- and dosage dependent. Taken together, we indicated that C42 can induce caspase-dependent apoptosis in AIPC cells, and the results presented here will broaden our knowledge about the molecular mechanisms by which C42 exerts its anticancer activity, and future work in this direction may provide valuable information in the development of these compounds into effective cancer therapeutic strategies against androgen-independent prostate cancer.

Silencing prion protein in MDA-MB-435 breast cancer cells leads to pleiotropic cellular responses to cytotoxic stimuli.

Prion protein (PrP) is well studied for its pathogenic role in prion disease, but its potential contribution to other pathological processes is less understood. PrP is expressed in a variety of cancers and at least in pancreatic and breast cancers, its expression appears to be associated with poor prognosis. To understand the role of PrP in breast cancer cells, we knocked down PrP expression in MDA-MB-435 breast cancer cells with small interfering RNA and subjected these cells to a series of analyses. We found that PrP knockdown in these cells does not affect cell proliferation or colony formation, but significantly influences the cellular response to cytotoxic stimuli. Compared to control cells, PrP knockdown cells exhibited an increased susceptibility to serum deprivation induced apoptosis, no change to staurosporine- or paclitaxel-induced cell deaths, and a reduced susceptibility to chemotherapy drug doxorubicin-induced cell death. To understand the mechanism of unexpected role of PrP in exacerbating doxorubicin-induced cytotoxicity, we analyzed cell death related Bcl-2 family proteins. We found that PrP knockdown alters the expression of several Bcl-2 family proteins, correlating with increased resistance to doxorubicin-induced cytotoxicity. Moreover, the enhanced doxorubicin resistance is independent of DNA damage related p53 pathway, but at least partially through the ERK1/2 pathway. Together, our study revealed that silencing PrP in MDA-MB-435 breast cancer cells results in very different responses to various cytotoxic stimuli and ERK1/2 signaling pathway is involved in PrP silencing caused resistance to doxorubicin.

Autophagy augmented by troglitazone is independent of EGFR transactivation and correlated with AMP-activated protein kinase signaling.

Troglitazone is a synthetic ligand of peroxisome proliferators activated receptor-gamma (PPARgamma) and induces apoptosis in a variety of malignant cells. However, the underlying mechanism of its regulatory role in macroautophagy (hereafter autophagy) remains largely unknown. Using fluorescence and electron microscopy, we observed that autophagosomes could be induced and identified upon troglitazone challenge in both primary and epidermal growth factor receptor (EGFR)-expressed porcine aortic endothelial (PAE) cells. We report here that troglitazone augments AMP-activated protein kinase-alpha (AMPKalpha) phosphorylation, reduces p70S6 kinase phosphorylation and stimulates autophagy that is independent of EGFR expression and transactivation. Troglitazone stimulus reduced neither lysosomal staining nor GFP-LC3 dots of HeLa cells, when the cells pretreated with AG1478, a specific EGFR kinase inhibitor. Furthermore, AG1478 additively enhanced the troglitazone-induced degradation of sequestosome 1 (SQSTM1/p62), which is a selective substrate of autophagy. Inhibition of AMPKalpha activity either by compound C or by RNA interference markedly reduced the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II), a good indicator of autophagy; whereas blockage of PPARgamma activity by the irreversible antagonist GW9662 or by overexpressing dominate-negative PPARgamma did not affect LC3-II accumulation and AMPK phosphorylation. Taken together, we demonstrate that autophagy promoted via troglitazone is correlated with AMPKalpha activation and independent of PPARgamma activation and EGFR transactivation.