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Several strains and species of lactic acid bacteria (LAB) are widely used in fermented foods, including dairy products and also as probiotics, because of their contribution to various health benefits in humans. Sortase enzymes decorate the bacterial cell wall with different surface proteins and pili for facilitating the interactions with host and environment for the colonization and beneficial effects. While the sortases and sortase anchored proteins from pathogens have been the prime focus of the research in the past, sortases from many non-pathogenic bacteria, including LAB strains, have attracted attention for their potential applications in vaccine delivery and other clinical interventions. Here, we report the purification and functional characterization of two sortases (housekeeping SrtA and pilus-specific SrtC) from a probiotic Lactococcus lactis. The purified sortases were found to be active against the putative LPXTG motif-based peptide substrates, albeit with differences. The in-silico analysis provides insights into the residues involved in substrate binding and specificity. Overall, this study sheds new light on the aspects of structure, substrate specificity, and function of sortases from non-pathogenic bacteria, which may have physiological ramifications as well as their applications in sortase-mediated protein bioconjugation.

The transcriptional repressor PARIS, a substrate of the ubiquitin E3 ligase parkin, represses the expression of the transcriptional co-activator, PGC-1α gene, and is involved in several pathological processes, including neurodegenerative disease and cancers. We have previously shown that SUMOylation of PARIS play an important role in its transcriptional repression activity. In addition, RNF4-mediated ubiquitination of SUMO2/3-conjugated PARIS is required for the control of PARIS-mediated transcriptional repression in HeLa cells that lack parkin expression. However, little is known about how PARIS ubiquitination and degradation are regulated in parkin-deficient cells. Here, we report that the deSUMOylase SENP3 interacted with PARIS and enhanced the ubiquitination of PARIS independently of its SUMOylation in HeLa cells. SENP3-enhanced PARIS ubiquitination mainly contributed to its proteasomal degradation, and required the oncogenic E3 ubiquitin ligase MDM2. MDM2 knockdown by small interfering RNA or expression of a dominant-negative MDM2 mutant inhibited the ubiquitination of PARIS. We further found that MDM2 activation via the PI3K/AKT pathway was involved in PARIS ubiquitination. Taken together, these results suggest that PARIS ubiquitination through SENP3-mediated MDM2 activation may control its functions in parkin-deficient cells.

The coronavirus 3C-like (3CL) protease is a Cysteine protease. It plays an important role in viral infection and immune escape by not only cleaving the viral polyprotein ORF1ab at 11 sites, but also cleaving the host proteins. However, there is still a lack of effective tools for determining the cleavage sites of the 3CL protease. This study systematically investigated the diversity of the cleavage sites of the coronavirus 3CL protease on the viral polyprotein, and found that the cleavage motif were highly conserved for viruses in the genera of Alphacoronavirus, Betacoronavirus and Gammacoronavirus. Strong residue preferences were observed at the neighboring positions of the cleavage sites. A random forest (RF) model was built to predict the cleavage sites of the coronavirus 3CL protease based on the representation of residues at cleavage site and neighboring positions by amino acid indexes, and the model achieved an AUC of 0.96 in cross-validations. The RF model was further tested on an independent test dataset composed of cleavage sites on host proteins, and achieved an AUC of 0.88 and a prediction precision of 0.80 when considering the accessibility of the cleavage site. Then, 1,079 human proteins were predicted to be cleaved by the 3CL protease by the RF model. These proteins were enriched in pathways related to neurodegenerative diseases and pathogen infection. Finally, a user-friendly online server named 3CLP was built to predict the cleavage sites of the coronavirus 3CL protease based on the RF model. Overall, the study not only provides an effective tool for identifying the cleavage sites of the 3CL protease, but also provides insights into the molecular mechanism underlying the pathogenicity of coronaviruses.

The capsid precursor (P1-2A) of foot-and-mouth disease virus is processed by the 3C protease (3Cpro) to VP0, VP3 and VP1 plus 2A. During capsid assembly, the VP0 is cleaved to VP4 plus VP2. Single amino acid changes in a conserved motif (YCPRP) near the C-terminus of VP1 can block processing of the capsid precursor by the 3Cpro, although the cleavage sites are located hundreds of amino acids distant from this motif, presumably due to misfolding. In contrast, we show here that the absence of the VP4 sequence during the synthesis of the capsid precursor does not affect its subsequent processing. Cleavage of this truncated precursor by 3Cpro at the VP3/VP1 and VP2/VP3 junctions occurred efficiently. Thus, in contrast to the presence of the YCPRP motif in VP1, there are no critical motifs near the N-terminus of the precursor, within VP4, required for correct cleavage by 3Cpro.

Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2Apro), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2Apro result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2Apro of genetic groups A and B hinder 2Apro as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2Apro cleave the substrate GAB1 at different sites, the 2Apro from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2Apro structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2Apro-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2Apro as a potential target for broad-spectrum anti-enteroviral inhibitors.

Genomic instability underlies genesis and the development of various types of cancer. During tumorigenesis, cancer initiating cells assume a set of features, which allow them to survive and proliferate. Different mutations and chromosomal alterations promote a selection of the most aggressive cancer clones that worsen the prognosis of the disease. Despite that caspase-2 was described as a protease fulfilling an initiator and an effector function in apoptosis, it has recently been discovered to play an important role in the maintenance of genomic integrity and normal chromosome configuration. This protein is able to stabilize p53 and affect the level of transcription factors, which activates cell response to oxidative stress. Here we focus on the discussion on the mechanism(s) of how caspase-2 regulates genomic stability and decreases tumorigenesis.

Pili of Gram-positive bacteria are flexible rod proteins covalently attached to the bacterial cell wall, that play important roles in the initial adhesion of bacterial cells to host tissues and bacterial colonization. Pili are formed by the polymerization of major and minor pilins, catalyzed by class C sortase (SrtC), a family of cysteine transpeptidases. The Gram-positive bacterium Clostridium perfringens has a major pilin (CppA), a minor pilin (CppB), and SrtC (CpSrtC). CpSrtC recognizes the C-terminal cell wall sorting signal motifs with five amino acid residues, LPSTG of CppA and LPETG of CppB, for the polymerization of pili. Here, we report biochemical analysis to detect the formation of Clostridium perfringens pili in vivo, and the X-ray structure of a novel intermolecular substrate-enzyme complex of CpSrtC with a sequence of LPST at the C-terminal site. The results showed that CpSrtC has a subsite for substrate-binding to aid polymerization of pili, and that the catalytic site has structural variations, giving insights into the enzyme catalytic reaction mechanism and affinities for the C-terminal cell wall sorting signal motif sequences.

Caspase-3 is the crucial executor caspase of apoptosis in mammalian cells, which is essential for chromatin condensation and DNA fragmentation. Although plants have no caspase-3 homologs, PBA1 acts as a plant caspase-3-like enzyme in plant programmed cell death (PCD). PCD occurs during the formation of secretory cavities in Citrus fruits; hence, secretory cavities could be utilized as a new cell biology model for investigating the regulatory mechanisms of plant PCD. To further study the association between PBA1 and PCD during secretory cavity development in Citrus fruits, CgPBA1 was identified in the fruit of Citrus grandis 'Tomentosa'. The temporal and spatial expression of CgPBA1 during secretory cavity development were analyzed using quantitative real-time PCR and in situ hybridization, and the morphological changes in the apoptotic cell nuclei were observed using TUNEL assay and ultra-thin section technology. The results revealed that the full-length cDNA of CgPBA1 contains a 711 bp ORF that encodes a putative protein containing 236 amino acid with a proteasome-β-6 functional domain that belongs to the Ntn hydrolase super family. CgPBA1 was predominantly expressed in the secretory cavities; its expression changes coincided with the morphological changes and DNA fragmentation in apoptotic cell nuclei. The green fluorescent fusion protein of CgPBA1 is also located in the nucleus of tobacco epidermal cells. Based on previous research and the findings of the present study, we speculate that CgPBA1 is a highly functional conserved protein in plants, and it might be involved in nuclear degradation during PCD for secretory cavity formation in C. grandis 'Tomentosa' fruits.

Liver sinusoidal endothelial cells (LSECs), which play a very critical role in liver regeneration, function in hypoxic environments, but few studies have elucidated the specific mechanism. As a hypoxia-sensitive gene, Sentrin/SUMO-specific protease 1(SENP1) is upregulated in solid tumors due to hypoxia and promotes tumor proliferation. We speculate that LSECs may upregulate SENP1 in hypoxic environments and that SENP1 may act on downstream genes to allow the cells to adapt to the hypoxic environment. To elucidate the reasons for the survival of LSECs under hypoxia, we designed experiments to explore the possible mechanism. First, we cultured murine LSECs in hypoxic conditions for a certain time (24 h and 72 h), and then, we observed that the proliferation ability of the hypoxia group was higher than that of the normoxia group, and the number of unique fenestrae of the LSECs in the hypoxia group was more than that of the LSECs in the normoxia group. Then, we divided the LSECs into several groups for hypoxic culture for time points (6 h, 12 h, 24 h, 36 h, and 72 h), and we found that the expression of SENP1, HIF-1α and VEGF was significantly upregulated. Then, we silenced SENP1 and HIF-1α with si-SENP1 and si–HIF–1α, respectively. SENP1, HIF-1α and VEGF were significantly downregulated, as determined by RT-PCR, WB and ELISA. Unexpectedly, the proliferation activity of the LSECs decreased and the fenestrae disappeared more in the si-SENP1 and si–HIF–1α groups than in the control group. It is concluded that LSECs cultured under hypoxic conditions may maintain fenestrae and promote proliferation through the SENP1/HIF-1α/VEGF signaling axis, thereby adapting to the hypoxic environment.

The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties. NT*-HRV3CP cleaves affinity tags at 4 °C in minutes, making it an attractive tool for the production of recombinant proteins for biotechnological, industrial and pharmaceutical applications.

The coronavirus disease 2019 (COVID-19) pandemic caused by 2019 novel coronavirus (2019-nCoV) has been a crisis of global health, whereas the effective vaccines against 2019-nCoV are still under development. Alternatively, utilization of old drugs or available medicine that can suppress the viral activity or replication may provide an urgent solution to suppress the rapid spread of 2019-nCoV. Andrographolide is a highly abundant natural product of the medicinal plant, Andrographis paniculata, which has been clinically used for inflammatory diseases and anti-viral therapy. We herein demonstrate that both andrographolide and its fluorescent derivative, the nitrobenzoxadiazole-conjugated andrographolide (Andro- NBD), suppressed the main protease (Mpro) activities of 2019-nCoV and severe acute respiratory syndrome coronavirus (SARS-CoV). Moreover, Andro-NBD was shown to covalently link its fluorescence to these proteases. Further mass spectrometry (MS) analysis suggests that andrographolide formed a covalent bond with the active site Cys145 of either 2019-nCoV Mpro or SARS-CoV Mpro. Consistently, molecular modeling analysis supported the docking of andrographolide within the catalytic pockets of both viral Mpros. Considering that andrographolide is used in clinical practice with acceptable safety and its diverse pharmacological activities that could be beneficial for attenuating COVID-19 symptoms, extensive investigation of andrographolide on the suppression of 2019-nCoV as well as its application in COVID-19 therapy is suggested., Highlights • 2019-nCoV and SARS-CoV main proteases exhibit similar enzyme kinetics. • Andrographolide inhibits enzyme activity of 2019-nCoV and SARS-CoV main proteases. • Andrographolide is covalently linked to active cysteine of 2019-nCoV main protease.

Background/Purpose Allergen-specific immunotherapy (SIT) is now considered curative to allergic diseases such as asthma. Mechanistically, our previous work showed DNA hypermethylation of cytokine genes, in T-helper cells, in allergic asthmatic children treated with allergen-SIT. In this study, we extended to work to assess possible changes in the DNA methylomes of peripheral blood mononuclear cells (PBMCs), isolated from mite allergen-SIT asthmatic children, to explore further the underlying methylation changes. Methods Thirteen allergic asthmatic children who received Der p-SIT, 12 non-SIT allergic asthmatic controls, and 12 healthy controls were enrolled. Bisulfite-converted DNA from Der p-stimulated PBMCs was analyzed using Human Methylation 450 k BeadChip. Pyrosequencing and quantitative real-time PCR were used to validate the DNA methylation levels and the gene expression of individual samples. Results We identified 108 significantly differentially methylated regions (DMRs) unique to Der p-treated PBMCs, with 53 probes linked to demethylated DMRs, and 55 probes linked to methylated DMRs. Three associated genes (BCL6, HSPG2, and HSP90AA1), of selected DMRs, were subjected to bisulfite pyrosequencing. Of these, BCL6 showed significant hypomethylation, while HSPG2 and HSP90AA1 were hypermethylated in SIT group, compared to the AA group. Furthermore, SIT group had significantly higher gene expression of BCL6 and lower gene expression of HSPG2. KEGG pathway analysis further revealed DMR genes involved in ECM-receptor interactions, asthma, and antigen processing and presentation pathways. Conclusions Several DNA regions showed DNA methylation altered by Der p specific immunotherapy, indicating desensitization-associated methylomes. Genes belonging to these SIT-altered pathways may represent therapeutic targets for better clinical management of asthma.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL−1, 116 μmole mg−1 min−1, 74.73 KJmol−1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high β–sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in β-sheet, and a significant increase in α-helix, suggesting a key role of β–sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

The COVID-19 pandemic caused by SARS-CoV-2 has emerged as a global catastrophe. The virus requires main protease for processing the viral polyproteins PP1A and PP1AB translated from the viral RNA. In search of a quick, safe and successful therapeutic agent; we screened various clinically approved drugs for the in-vitro inhibitory effect on 3CLPro which may be able to halt virus replication. The methods used includes protease activity assay, fluorescence quenching, surface plasmon resonance (SPR), Thermofluor® Assay, Size exclusion chromatography and in-silico docking studies. We found that Teicoplanin as most effective drug with IC50 ~ 1.5 μM. Additionally, through fluorescence quenching Stern–Volmer quenching constant (KSV) for Teicoplanin was estimated as 2.5 × 105 L·mol−1, which suggests a relatively high affinity between Teicoplanin and 3CLPro protease. The SPR shows good interaction between Teicoplanin and 3CLPro with KD ~ 1.6 μM. Our results provide critical insights into the mechanism of action of Teicoplanin as a potential therapeutic against COVID-19. We found that Teicoplanin is about 10–20 fold more potent in inhibiting protease activity than other drugs in use, such as lopinavir, hydroxychloroquine, chloroquine, azithromycin, atazanavir etc. Therefore, Teicoplanin emerged as the best inhibitor among all drug molecules we screened against 3CLPro of SARS-CoV-2., Highlights • 3CLPro protease is an attractive drug target to halt the viral replications of SARS-CoV-2. • Screening of approved molecules against 3CLPro protease of SARS-CoV-2 • Biochemical activity and biophysical analysis of molecules against 3CLPro protease • Teicoplanin found to be an effective inhibitor of 3CLPro protease of SARS-CoV-2

Background House dust mites (HDMs) are among the most important allergen sources containing many different allergenic molecules. Analysis of patients from a double-blind, placebo-controlled allergen-specific immunotherapy (AIT) study indicated that patients may benefit from AIT to different extents depending on their molecular sensitization profiles. Objective Our aim was to investigate in a real-life setting whether stratification of patients with HDM allergy according to molecular analysis may enhance AIT success. Methods Serum and nasal secretion samples from patients with HDM allergy (n = 24) (at baseline, 7, 15, 33, and 52 weeks) who had received 1 year of treatment with a well-defined subcutaneous AIT form (Alutard SQ 510) were tested for IgE and IgG reactivity to 15 microarrayed HDM allergen molecules with ImmunoCAP Immuno-solid-phase Allergen Chip technology. IgG subclass levels to allergens and peptides were determined by ELISA, and IgG blocking was assessed by basophil activation. In vitro parameters were related to reduction of symptoms determined by combined symptom medication score and visual analog scale score. Results Alutard SQ 510 induced protective IgG mainly against Dermatophagoides pteronyssinus (Der p) 1 and Der p 2 and to a lesser extent to Der p 23, but not to the other important allergens such as Der p 5, Der p 7, and Der p 21, showing better clinical efficacy in patients sensitized only to Der p 1 and/or Der p 2 as compared with patients having additional IgE specificities. Conclusion Stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.

Pseudogenes, which are long noncoding RNAs that originate from protein-coding genes, have been suggested to play important roles in disease. Although studies have revealed high expression of legumain (LGMN) in many types of tumors, the regulation of LGMN remains largely unknown. Here, we found that a novel LGMN pseudogene (LGMNP1) was upregulated in glioblastoma (GBM) tissues and high LGMNP1 expression in GBM cells enhanced proliferation and invasion. Biochemical analysis showed that cytoplasmic LGMNP1 functionally targeted miR-495-3p in a manner involving an RNA-induced silencing complex. Dual-luciferase reporter assays demonstrated that LGMN was a target of miR-495-3p, and LGMN was upregulated and positively correlated with LGMNP1 in GBM. Moreover, miR-495-3p was downregulated and negatively correlated with LGMNP1 in GBM tissues. Notably, the tumor-promoting effects of LGMNP1 upregulation could be alleviated by miR-495-3p mimics. Furthermore, GBM cells overexpressing LGMNP1 exhibited more aggressive tumor progression and elevated LGMN expression in vivo. Thus, our data illustrate that LGMNP1 exerts its oncogenic activity, at least in part, as a competitive endogenous RNA (ceRNA) that elevates LGMN expression by sponging miR-495-3p. CeRNA-mediated miRNA sequestration might be a novel therapeutic strategy in GBM.

Chikungunya virus; the pathogen for chikungunya febrile and arthritic disease, having 11.8 kb positive-sense RNA genome encodes polyproteins for structural and non-structural regions. The polyprotein (P1234) corresponding to the non-structural part from 5′ end gets auto-cleaved by the action of nsP2 protease, which leads to the generation of individual functional enzymatic proteins like nsP4, nsP1, nsP2 and nsP3. Thus, nsP2 protein initiates viral replication. Targeting nsP2 to block virus replication has always been the foremost strategy to develop antivirals. Plant-based molecules are one of the top choices to develop as inhibitor due to their less toxicity and wide availability. Using a combination of receptor-based docking and MD simulations, we identified a flavanone glycoside- naringin, which binds to nsP2 protease at nM affinity. The biomolecular interaction between naringin and nsP2 was established through SPR. As discerned through FTIR and intrinsic fluorescence studies, upon binding with naringin, a global structural change in nsP2 occurs. This structural modulation in nsP2 due to binding of naringin is likely to interfere with the normal functioning of this enzyme during the viral life cycle. In conclusion, this report highlights the potential of naringin as an anti-viral agent against Chikungunya.

Background The rapidly enlarging COVID-19 pandemic caused by the novel SARS-corona virus-2 is a global public health emergency of an unprecedented level. Unfortunately no treatment therapy or vaccine is yet available to counter the SARS-CoV-2 infection, which substantiates the need to expand research efforts in this direction. The indispensable function of the main protease in virus replication makes this enzyme a promising target for inhibitors screening and drug discovery to treat novel coronavirus infection. The recently concluded α-ketoamide ligand-bound X-ray crystal structure of SARS-CoV-2 Mpro (PDB ID: 6Y2F) from Zhang et al. has revealed the potential inhibitor binding mechanism and the molecular determinants responsible for substrate binding. Methods For the study, we have targeted the SARS-CoV-2 Mpro for the screening of FDA approved antiviral drugs and carried out molecular docking based virtual screening. Further molecular dynamic simulation studies of the top three selected drugs carried out to investigated for their binding affinity and stability in the SARS-CoV-2 Mpro active site. The phylogenetic analysis was also performed to know the relatedness between the SARS-CoV-2 genomes isolated from different countries. Results The phylogenetic analysis of the SARS-CoV-2 genome reveals that the virus is closely related to the Bat-SL-CoV and does not exhibit any divergence at the genomic level. Molecular docking studies revealed that among the 77 drugs, screened top ten drugs shows good binding affinities, whereas the top three drugs: Lopinavir–Ritonavir, Tipranavir, and Raltegravir were undergone for molecular dynamics simulation studies for their conformational stability in the active site of the SARS-CoV-2 Mpro protein. Conclusions In the present study among the library of FDA approved antiviral drugs, the top three inhibitors Lopinavir–Ritonavir, Tipranavir, and Raltegravir show the best molecular interaction with the main protease of SARS-CoV-2. However, the in-vitro efficacy of the drug molecules screened in this study further needs to be corroborated by carrying out a biochemical and structural investigation.

Ebselen is an organoselenium compound exhibiting hydroperoxide- and peroxynitrite-reducing activity, acting as a glutathione peroxidase and peroxiredoxin enzyme mimetic. Ebselen reacts with a multitude of protein thiols, forming a selenosulfide bond, which results in pleiotropic effects of antiviral, antibacterial and anti-inflammatory nature. The main protease (Mpro) of the corona virus SARS-CoV-2 is a potential drug target, and a screen with over 10,000 compounds identified ebselen as a particularly promising inhibitor of Mpro (Jin, Z. et al. (2020) Nature 582, 289–293). We discuss here the reaction of ebselen with cysteine proteases, the role of ebselen in infections with viruses and with other microorganisms. We also discuss effects of ebselen in lung inflammation. In further research on the inhibition of Mpro in SARS-CoV-2, ebselen can serve as a promising lead compound, if the inhibitory effect is confirmed in intact cells in vivo. Independently of this action, potential beneficial effects of ebselen in COVID-19 are ascribed to a number of targets critical to pathogenesis, such as attenuation of inflammatory oxidants and cytokines., Graphical abstract Image 1, Highlights • Ebselen, an organoselenium compound, exhibits anti-inflammatory and antiviral activity. • Ebselen is a glutathione peroxidase and peroxiredoxin mimetic. • Ebselen reacts with a multitude of protein thiols, resulting in pleiotropic effects. • Ebselen is an effective inhibitor of Mpro, the main protease of SARS-CoV-2. • Ebselen may serve as lead compound for drugs targeting COVID-19.

Purpose Membrane engineering has versatile applications in adoptive cell therapies, immune therapy or drug delivery. Incorporation of lipidated antibody-derived ligands into cells may enforce supraphysiological cell interactions that offer new therapeutic approaches. A challenge is the defined synthesis of lipidated ligands that effectively interact with such membranes. Methods Sortase-A was used to attach a PEGylated, dimyristyl lipid-anchor on single-domain antibodies (VHH). The membrane insertion was investigated on liposomal bilayers, myeloid-derived suppressor cells (MDSC) and T cells. Results The lipidated VHHs remodeled liposomal as well as cellular membranes. The VHH carrying liposomes were successfully targeted towards antigen-positive cells. MDSC and T cells were both modified with lipidated VHHs as detected with an FITC-anti-llama antibody. T cells that carried an anti-CD11b VHH showed cellular association in vitro with CD11b+Gr-1+ MDSC in a two-dimensional magnetic activated cell sorting / flow-cytometry assay. Conclusion The applied combination of chemoenzymatic ligation, PEGylated lipid anchors and single-domain antibodies delivers water-soluble and chemically defined lipidated ligands, which readily associate with liposomal and cellular membranes. This enables liposomal drug targeting and artificial cell-cell interactions. Hence, the presented concept for lipidation of single-domain antibodies is promising for further application in the field of drug delivery or cell-based therapies.

Oral chemotherapy offers a more convenient treatment option for cancer patients but the effectiveness is significantly hindered by the limited drug delivery efficiency. Herein, we designed legumain-activable melittin (LM) decorated polymeric nanovehicles loading IR-780 and sorafenib (LPN) to enhance their oral delivery to tumors, with efficient accumulation and penetration capacity, for combinational photothermal-chemotherapy of gastric cancer. The nanosized LPN displayed good stability in simulated gastrointestinal fluids. After oral administration, the oral bioavailability of sorafenib was remarkably improved (75.9-fold by LPN versus free drug suspension). Moreover, the orally administered LPN could preferentially accumulate at the tumor site and penetrate into the interior regions of the tumor mass. Upon combination with laser irradiation, LPN produced notable inhibition of tumor growth, which was more effective than the counterpart unmodified nanovehicles. Therefore, LPN represents an encouraging oral delivery nanoplatform with favorable tumor accumulation and penetration capability for oral combinational cancer therapy.

Bean common mosaic virus (BCMV) causes severe disease in Phaseolus vulgaris plants. One of its non structural protein, the helper-component proteinase (HcPro) involves in multiple roles in aphid transmission, RNA binding, suppression of gene silencing and protease activity. The multifunctional role of HcPro hint towards its regulation at multiple host cellular sites. The mechanisms of these regulatory activities are poorly understood. Therefore, it is very important to study the molecular level interaction of HcPro with different cellular components. In this study, we demonstrate that the HcPro interacts with RuBisCo, an enzyme of chloroplast origin which might plays a crucial role in virus infection. A further line of experiments were carried out with factors of nuclear origin. Due to nucleic acid binding activity of HcPro, it showed interaction with dsDNA of nucleosome, as ascertained through electrophoretic mobility shift assay (EMSA). Interestingly, HcPro interacts with host nucleoprotein histones, H3 and H4. The gel-overlay assay and native electrophoresis-western blot analysis (NEWeB) revealed a direct interaction of BCMV HcPro with host nucleosome and with histones. These findings suggest that the BCMV through HcPro, not only utilize the host cytoplasmic components but also use host nuclear factors for its propagation and disease development.

Background The cell membrane-derived initiators of coagulation, tissue factor (TF) and anionic phospholipid (aPL), are constitutive on the herpes simplex virus type 1 (HSV1) surface, bypassing physiological regulation. TF and aPL accelerate proteolytic activation of factor (F) X to FXa by FVIIa to induce clot formation and cell signaling. Thus, infection in vivo is enhanced by virus surface TF. HSV1-encoded glycoprotein C (gC) is implicated in this tenase activity by providing viral FX binding sites and increasing FVIIa function in solution. Objective To examine the biochemical influences of gC on FVIIa-dependent FX activation. Methods Immunogold electron microscopy (IEM), kinetic chromogenic assays and microscale thermophoresis were used to dissect tenase biochemistry. Recombinant TF and gC were solubilized (s) by substituting the transmembrane domain with poly-histidine, which could be orientated on synthetic unilamellar vesicles containing Ni-chelating lipid (Ni-aPL). These constructs were compared to purified HSV1 TF±/gC ± variants. Results IEM confirmed that gC, TF, and aPL are simultaneously expressed on a single HSV1 particle where the contribution of gC to tenase activity required the availability of viral TF. Unlike viral tenase activity, the cofactor effects of sTF and sgC on FVIIa was additive when bound to Ni-aPL. FVIIa was found to bind to sgC and this was enhanced by FX. Orientation of sgC on a lipid membrane was critical for FVIIa-dependent FX activation. Conclusions The assembly of gC with FVIIa/FX parallels that of TF and may involve other constituents on the HSV1 envelope with implications in virus infection and pathology.

Engineering and bioconjugation of proteins is a critically valuable tool that can facilitate a wide range of biophysical and structural studies. The ability to orthogonally tag or label a domain within a multidomain protein may be complicated by undesirable side reactions to noninvolved domains. Furthermore, the advantages of segmental (or domain-specific) isotopic labeling for NMR, or deuteration for neutron scattering or diffraction, can be realized by an efficient ligation procedure. Common methods—expressed protein ligation, protein trans-splicing, and native chemical ligation—each have specific limitations. Here, we evaluated the use of different variants of Staphylococcus aureus sortase A for a range of ligation reactions and demonstrate that conditions can readily be optimized to yield high efficiency (i.e. completeness of ligation), ease of purification, and functionality in detergents. These properties may enable joining of single domains into multidomain proteins, lipidation to mimic posttranslational modifications, and formation of cyclic proteins to aid in the development of nanodisc membrane mimetics. We anticipate that the method for ligating separate domains into a single functional multidomain protein reported here may enable many applications in structural biology.

Protein antigens are, in general, weakly immunogenic, and therefore require co-delivery with adjuvants to stimulate potent immune responses. The fusion of (poly)peptide antigens to immunostimulatory adjuvants (e.g. Toll-like receptor (TLR) agonists) has been demonstrated to greatly improve vaccine potency compared to mixtures of antigen and adjuvant. Chemical approaches, to enable the rapid, site-specific and high-yielding linkage of TLR2 ligands to recombinant protein antigens, have been previously optimized. These approaches require the use of denaturing conditions to ensure high reaction yields, which limits their application, as maintenance of native protein folding is necessary to elicit antibodies against conformational epitopes. Here, this work aimed to optimize an alternative method, to ensure the efficient bioconjugation of TLR2 ligands onto folded protein antigens. An enzyme-mediated approach, using Staphylococcus aureus sortase A (or a penta mutant with enhanced efficiency), was optimized for reaction yield and time, as well as enzyme type and amount. This approach enabled the site-specific conjugation of the TLR2-agonist fibroblast-stimulating lipopeptide-1 (FSL-1) onto a model group A Streptococcus (GAS) recombinant polytope antigen under conditions that maintain protein folding, yielding a homogeneous, molecularly-defined product, with ligation yields as high as 90%. Following intramuscular (IM) administration of the ligation product to humanized plasminogen AlbPLG1 mice, high-titer, antigen-specific IgG antibodies were observed, which conferred protection against subcutaneous challenge with GAS strain 5448. In comparison, mixtures of the GAS antigen with aluminum hydroxide or FSL-1 failed to provide protection, with the FSL-1 mixture yielding ~1000-fold lower antigen-specific IgG antibody titers, and the mixture with alum yielding a Th2-biased response compared to the more balanced Th1/Th2 responses observed with the FSL-1 conjugate. Overall, a FSL-1 bioconjugation method for the efficient production of antigen-TLR2 agonist conjugates, which maintain protein folding, was produced, with broad utility for the development of self-adjuvanting vaccines against subunit protein antigens.

Ananain (EC 3.4.22.31) accounts for less than 10% of the total enzyme in the crude pineapple stem extract known as bromelain, yet yields the majority of the proteolytic activity of bromelain. Despite a high degree of sequence identity between ananain and stem bromelain, the most abundant bromelain cysteine protease, ananain displays distinct chemical properties, substrate preference and inhibitory profile compared to stem bromelain. A tripeptidyl substrate library (REPLi) was used to further characterize the substrate specificity of ananain and identified an optimal substrate for cleavage by ananain. The optimal tripeptide, PLQ, yielded a high kcat/Km value of 1.7 x 106 M−1s−1, with cleavage confirmed to occur after the Gln residue. Crystal structures of unbound ananain and an inhibitory complex of ananain and E−64, solved at 1.73 and 1.98 A, respectively, revealed a geometrically flat and open S1 subsite for ananain. This subsite accommodates diverse P1 substrate residues, while a narrow and deep hydrophobic pocket-like S2 subsite would accommodate a non-polar P2 residue, such as the preferred Leu residue observed in the specificity studies. A further illustration of the atomic interactions between E−64 and ananain explains the high inhibitory efficiency of E−64 toward ananain. These data reveal the first in depth structural and functional data for ananain and provide a basis for further study of the natural properties of the enzyme.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the pathogen that caused the global COVID-19 outbreak. The 3C-like protease (3CLsuppro/sup) of SARS-CoV-2 plays a key role in virus replication and has become an ideal target for antiviral drug design. In this work, we have employed bioluminescence resonance energy transfer (BRET) technology to establish a cell-based assay for screening inhibitors against SARS-CoV-2 3CLsuppro/sup, and then applied the assay to screen a collection of known HIV/HCV protease inhibitors. Our results showed that the assay is capable of quantification of the cleavage efficiency of 3CLsuppro/supwith good reproducibility (Z' factor is 0.59). Using the assay, we found that 9 of 26 protease inhibitors effectively inhibited the activity of SARS-CoV-2 3CLsuppro/supin a dose-dependent manner. Among them, four compounds exhibited the ability to bind to 3CLsuppro/supin vitro. HCV protease inhibitor simeprevir showed the most potency against 3CLsuppro/supwith an ECsub50/subvale of 2.6 μM, bound to the active site pocket of 3CLsuppro/supin a predicted model, and importantly, exhibited a similar activity against the protease containing the mutations P132H in Omicron variants. Taken together, this work demonstrates the feasibility of using the cell-based BRET assay for screening 3CLsuppro/supinhibitors and supports the potential of simeprevir for the development of 3CLsuppro/supinhibitors.

The Ubiquitin-fold modifier 1 (Ufm1) is a ubiquitin-like protein that can also be conjugated to protein substrates and subsequently alter their fates. Both UFMylation and de-UFMylation are mediated by Ufm1-specific proteases (UFSPs). In humans, it is widely believed that UFSP2 is the only active Ufm1 protease involved in Ufm1 maturation and de-UFMylation, whereas UFSP1 is thought to be inactive. Here, Liang et al. provide strong evidence showing that human UFSP1 is also an active Ufm1 protease. These results solve an age-old mystery in the human Ufm1 conjugation system and could have a greater impact not only on Ufm1 biology but also on the translation of genes employing nontraditional start codons.

Purification of native gingipains is challenging because these proteases are frequently associated with the cell surface, which affects yield. This study aimed to purify native Arg-gingipain (RgpA) from Porphyromonas gingivalis Outer Membrane Vesicles (OMV).Native RgpA was purified from P. gingivalis strain ATCC33277 OMV using a strategy including ultracentrifugation, sonication, and successive anionic and cationic fast protein liquid chromatography (FPLC). The presence and purity of the protease were confirmed by SDS-PAGE and detection of protease activity using fluorogenic substrates. Rat antibodies produced against the unique adhesin hemagglutinin (H1) domain of RgpA (amino acids 719-865) were titrated by ELISA at a 1:100 dilution using whole P. gingivalis lysate as an antigen and western blotting to detect a 75 kDa band corresponding to RgpA.Double anionic-cationic FLPC yielded prominent peaks with evident amidolytic gingipain activity of the appropriate molecular weight, as confirmed by western blotting. The final RgpA yield from 1 L of bacterial culture with colony forming unit (CFU) (LogThis protocol allows purification of native RgpA from OMV that retains protease activity.

Actinidin, a cysteine protease in green kiwifruit (Actinidia deliciosa), has been identified as a potential enzyme to hydrolyse gluten within the lumen of the gastrointestinal tract (GIT). The present study aimed to further evaluate the effect of purified actinidin sourced from green kiwifruit on the digestion of gluten and the release of immunogenic peptides during GIT digestion using an in vitro semi-dynamic GIT digestion model. Purified gluten was digested for 180 min with or without actinidin and subsequently analysed for free amino groups (o-phthaldialdehyde) to determine the degree of hydrolysis (DH), gluten R5 epitopes (ELISA), and peptide profiles (mass spectrometry). Strong interactions were observed between treatment (GIT digestion with or without actinidin) and digestion time for the DH of gluten (P 0.01), amount of free amino groups released into the small intestine (P 0.01), and amount of gluten epitopes present in the small intestine (P 0.001). The rate of increase of DH of gluten and the amount of R5 epitopes present in the small intestine during the first 30 min of GIT digestion with actinidin was 0.3%/min and 4.8 ng/g of gluten respectively, whereas it was 0.01%/min and 60.9 ng/g of gluten respectively without actinidin. These results were corroborated by untargeted peptidomics, with a 1.5-fold lower number of known immunogenic epitopes reaching the small intestine at 30 min of GIT digestion when actinidin was present compared to the control. Present results demonstrate that actinidin enhanced the rate of proteolysis of gluten and reduced the number of immunogenic gluten epitopes reaching the small intestine during simulated semi-dynamic GIT digestion.

The discovery of antiviral agents against SARS-CoV-2 is an important step toward ending the COVID-19 pandemic and to tackle future outbreaks. In this context, the main protease (M

SARS-CoV-2 has raised the alarm to search for effective therapy for this virus. To date several vaccines have been approved but few available drugs reported recently still need approval from FDA. Remdesivir was approved for emergency use only. In this report, the SARS-CoV-2 3CLpro was expressed and purified. By using a FRET-based enzymatic assay, we have screened a library consisting of more than 300 different niclosamide derivatives and identified three molecules JMX0286, JMX0301, and JMX0941 as potent allosteric inhibitors against SARS-CoV-2 3CLpro, with IC

The aim was to determine the changes of inflammatory mediator expression in human macrophages stimulated with outer membrane vesicles purified from Porphyromonas gingivalis.outer membrane vesicles purified by ultracentrifugation from ATCC 33277 and W83 P. gingivalis strains were used for stimulating human macrophages and determine their inflammatory mediator expression changes. U937 monocyte cells line were differentiated into macrophages and stimulated with outer membrane vesicles for 30 min and six hours. In Independent experiments, the outer membrane vesicles and viable bacteria control were pre-treated with the gingipain inhibitors KYT-1 and KYT-36 (Arg-gingipain and Lys-gingipain, respectively) or Polymyxin-B to block the lipopolysaccharide activity to evaluate the secretion changes of immune mediators IL-1β, IL-6, TNF-α, IL-8, MCP-1, MIP-1α and RANTES by flow cytometry. A factorial ANOVA was used to analyze the data.The outer membrane vesicles of P. gingivalis ATCC 33277 displayed higher Arg-gingipain activity than those obtained from the P. gingivalis W83 strain (0.6 U/μg vs. 0.46 U/μg). Although the outer membrane vesicles of P. gingivalis stimulated the production of cytokines and chemokines, specific Arg-gingipain and Lys-gingipain inhibition induced significant increases in IL-1β, IL-6, IL-8, MCP-1, and RANTES levels, and this induction was significantly greater at 6 h compared to 30 min (*p 0.05). On the contrary, TNF-α secretion decreased when gingipains were blocked.outer membrane vesicles may play a dual role during P. gingivalis infection based on their ability to induce changes in the immune responses of human macrophages, probably via gingipain-dependent events.

Parasitic cysteine proteases such as rhodesain (TbCatL) from Trypanosoma brucei rhodesiense are relevant targets for developing new potential drugs against parasitic diseases (e. g. Human African Trypanosomiasis). Designing selective inhibitors for parasitic cathepsins can be challenging as they share high structural similarities with human cathepsins. In this paper, we describe the development of novel peptidomimetic rhodesain inhibitors by applying a structure-based de novo design approach and molecular docking protocols. The inhibitors with a new scaffold in P2 and P3 position display high selectivity towards trypanosomal rhodesain over human cathepsins L and B and high antitrypanosomal activity. Vinylsulfonate 2a has emerged as a potent rhodesain inhibitor (k

Human osteosarcoma is the most common primary cancer of the bone. Multiple mechanisms underlying cell growth, apoptosis, bone development, and drug resistance are important in the development of osteosarcoma therapy, which remains to be fully studied.We collected thirty-paired tumor tissues and the adjacent normal ones from osteosarcoma patients. Two osteosarcoma cell lines (SAOS2, U2OS) were used for in vitro experiments. RT-qPCR and Western blot were used for gene expression detection. We applied starBase to predict the potential binding sites. Then, the luciferase reporter assay was used to confirm the potential direct interaction. Besides, we applied CCK-8, EdU assay, and flow cytometric assays to detect cell growth and apoptosis rate. We used wound healing and transwell assays to determine cell migration and invasion abilities. Additionally, we constructed a cisplatin-resistant osteosarcoma cell line to study the potential impact of the regulatory axis on drug resistance.Small nucleolar RNA host gene 16 (SNHG16) and autophagy-related 4B (ATG4B) were significantly upregulated in osteosarcoma tissues than the normal ones, and the higher expression level of SNHG16 predicted a poor prognosis in osteosarcoma patients. By contrast, the expression level of miR-16 was markedly lower in tumor tissues and was negatively correlated with SNHG16 (p 0.001). SNHG16 was shown to promote cell growth, migration, and invasion, while miR-16 reversed this impact. Meanwhile, overexpression of ATG4B significantly promoted the development of osteosarcoma cells attenuated by SNHG16 knockdown or miR-16 mimics. Specifically, overexpression of ATG4B promoted cisplatin-induced autophagy and inhibited cell apoptosis rate, which enhanced the cisplatin resistance in osteosarcoma cell lines.Overall, our findings showed the importance of the regulatory axis of SNHG16/miR-16/ATG4B underlying osteosarcoma progression and chemoresistance to cisplatin. This research would benefit the therapy development in the treatment of osteosarcoma.

Zona occludens-1 (ZO-1) is a key component of tight junctions that govern the function of the endothelial barrier against tumor metastasis. Factors secreted by tumor cells contribute to the maintenance of tumor vascular networks. How tumor cell-derived protein signals regulate ZO-1 expression is unclear. Here, we explored the effect of tumor cell-secreted asparaginyl endopeptidase (AEP) on the permeability of endothelial cells in the tumor microenvironment. First, we confirmed the existence of AEP in conditioned medium (CM) from AEP-overexpressing MDA-MB-231 and 4T1 cells. Treatment with CM from AEP-overexpressing tumor cells increased the permeability and tumor cell transversal of an endothelial monolayer. Furthermore, CM from AEP-overexpressing tumor cells suppressed endothelial ZO-1 expression, as well as ZO-1-associated nucleic acid binding protein ZONAB. In addition, the level of phosphorylated STAT3 was increased by treatment with AEP-containing CM. A mutation of RGD or blocking integrin αvβ3 with antibody recovered the ZO-1 downregulation induced by AEP. In vivo, a lung metastatic mouse model showed increased endothelial permeability in the AEP-overexpressing group compared with the control group. An orthotopic tumor transplantation model was established using AEP-overexpression and compared with mice receiving control 4T1 cells. Compared with controls, overexpression of AEP increased lung metastatic foci and area, as well as vascular instability in primary tumors or lung metastatic sites. Moreover, endothelial ZO-1 was decreased in the AEP-overexpressing group. Taken together, our data show that tumor cell-derived AEP increases the permeability of endothelial barriers. Interactions between RGD and endothelial integrin αvβ3 mediate this effect by downregulating ZO-1.

The vacuolar processing enzyme (VPE) plays an important role in PCD and was originally identified as the proteinase responsible for the maturation and activation of vacuolar proteins in plants. We found that γVPE is involved in PCD of xylem fiber cells through the activation of CEP1 proproteases into mature protease in Arabidopsis. The γVPE protein was expressed specifically in cambium cells cambium, the primary phloem and the primary xylem during stem development. The recombinant γVPE appearing as a proenzyme at pH 7.0, and then transforming into a 40-kD mature enzyme at pH 5.5 in vitro by self-cleaving. The mature γVPE protein activated CEP1 maturation in vitro, whereas this activity was inhibited in the γvpe mutant. Transmission electron microscopy showed delayed PCD in fiber cells and thickening of secondary fiber cell walls in the γvpe mutant. Transcriptome analysis showed that the expression of 2001 genes was significantly altered expression in the γvpe mutants, and most of them are important for secondary cell wall formation and PCD. Our results demonstrate that γVPE is a crucial processing enzyme for xylem fiber cells PCD during stem development.

Falcipain-2(FP2), a cysteine protease from Plasmodium falciparum, cleaves host erythrocyte hemoglobin and specific membrane skeleton components during the parasite life cycle. Therefore its inhibition has been considered as an attractive approach to combat the disease. SerpinB3 (SPB3) belongs to the ovalbumin-serpin family and is a potent cross-class inhibitor of cysteine cathepsins L, K, S and papain. This study explored the possibility of inhibition of FP2 by SPB3. It turned out that general proteolytic activities as well as specific hemoglobinolytic activity of FP2 have been inhibited by SPB3. Furthermore, studies have been designed to investigate and characterize the mechanism of inhibition in comparison with proteases Cathepsin L (CTSL) and papain. The Ki value of inhibition for FP2, measured against its specific substrate (VLK-pNA), is 338.11 nM and stoichiometry (I/E ratio) of inhibition is 1. These values are comparable to CTSL and papain. Analytical gel filtration profile and CD spectroscopy data confirm FP2-SPB3 complex formation. Our studies revealed that interaction of SPB3 with FP2 is non-covalent type like that of CTSL and papain but unlike other serine protease-inhibiting serpins. An in-silico docking and simulation study have been performed with FP2 as well as CTSL and results suggest different binding mode for FP2 and CTSL, though both the complexes are stable with significant contribution from electrostatic energy of interaction. We further showed a disease state mutant SPB3-Gly351Ala performed better anti-protease activity against FP2. This study, for the first time, has shown a serpin family inhibitor from human could efficiently inhibit activity of FP2.

Sortases (Srts) are transpeptidase enzymes that anchor plethora of surface proteins with LPXTG and NPQTN motifs to the cell wall of Gram-positive bacteria, and they are potential targets for the development of anti-infective agents. Despite the existence of numerous inhibitors for Staphylococcus aureus SrtA (SaSrtA), only two inhibitors are known for Staphylococcus aureus SrtB (SaSrtB). Moreover, no study has yet documented the anti-virulence potential of cone snail venom conopeptides against these virulence factors. Here we report M2-conotoxin and contryphan-R as effective inhibitory agents that may competitively block the binding of natural substrates with SaSrtA and SrtB, respectively, via molecular docking and dynamic simulation assays. M2-conotoxin also exhibited strong binding inside the catalytic grooves of distantly related SrtA homologs from Streptococcus mutans (SmSrtA) and Streptococcus agalactiae (SgSrtA). On the other hand, contryphan-R failed to occupy the substrate binding site of closely related Bacillus anthracis SrtB (BaSrtB), but successfully blocked the catalytic site of very divergent C. Perfringens SrtB (CpSrtB). Hence, these naturally existing venom peptides and their mimetics may serve as promising candidates for further development of therapeutically useful anti-infectives for the treatment of infections caused by multi-drug resistant bacterial pathogens having SrtA and SrtB in their enzymatic set.

Introduction Bleomycin pulmonary toxicity (BPT) is a potentially life-threatening consequence of bleomycin usage in patients. An overproduction of epithelium-derived cytokines, habitually linked to allergic inflammation, has been recently revealed in experimental models of BPT. Methods We assessed retrospectively our cohort of patients with Hodgkin Lymphoma treated with bleomycin between 2014 and 2016 for their demographic, clinical features, including BPT development, atopy status and risk factors for BPT. Then they were invited for allergy testing and blood sample collection. The samples were stimulated with different stimuli (Bleomycin, IL-33, TSLP) for 24 h on cell culture. The culture supernatants were analysed for TGF-β, Galectin3, Arginin, Amphiregulin, Eotaxin, IFNγ, TNFα, IL1β, 4, 5, 6, 10, 13, 17, MIP-1α, and bleomycin hydrolase (BLH) levels. Results The cohort consisted of 51 patients showed that atopy was the only significant risk factor for BPT occurrence (OR: 7.2, p = 0.007). Fourteen subjects were included for blood analysis. The analysis of supernatants at the unstimulated condition revealed that BLH and Amphiregulin were significantly lower in patients who had BPT than controls. The BLH cut-off that best identified a history of BPT was 175.31 (Sensitivity: 62.5%, specificity: 100%). Following the stimulation, BLH reduced compared to the unstimulated condition and the difference between groups remained significant (p Conclusion Our study is the first to report that low levels of bleomycin hydrolase in allergic individuals may be predisposing to a possible pathway of fibrosis.

Doxorubicin is a chemotherapeutic agent commonly used to treat breast cancer. However, breast cancer often develops drug resistance, leading to disease recurrence and poor prognosis. Delineating the mechanisms underlying drug resistance is imperative for overcoming the challenge of treating doxorubicin-resistant breast cancer. In this study, by identifying the possible role of Sentrin/SUMO-specific proteases (SENPs) in doxorubicin resistance, we show here that among the 6 members of SENPs, only SENP2 is downregulated in doxorubicin-resistant MCF-7 (MCF-7/adr) and MDA-MB-231 (dr) breast cancer cells, as compared with sensitive counterparts. In addition, functionally, SENP2 overexpression resensitizes resistant breast cancer cells to doxorubicin treatment, and its knockdown confers doxorubicin resistance in sensitive ones. Moreover, NF-κB pathway is activated in MCF-7/adr cells, however, treatment with Bay 11-7085, one specific inhibitor of this pathway, reverses resistance to doxorubicin, suggesting that NF-κB pathway activation contributes to doxorubicin resistance in MCF-7/adr cells. We further show that SENP2 overexpression enhances NEMO deSUMOylation and suppresses NF-κB activation particularly in MCF-7/adr cells. Furthermore, SENP2 overexpression-induced sensitivity of MCF-7/adr cells to doxorubicin is drastically abrogated when treated with NF-κB pathway activator, thus establishing a causal link between SENP2-suppressed NF-κB pathway and enhanced doxorubicin sensitivity in breast cancer cells. Overall, this study reveals a novel function of SENP2 in counteracting doxorubicin resistance in breast cancer, and highlights the critical role of NF-κB suppression in mediating this effect.

This study investigated the expression and role of immunoproteasome (i-proteasome) in a cell model of Parkinson's disease (PD). The cytotoxicity of rotenone was measured by CCK-8 assay. The i-proteasome β1i subunit PSMB9 was suppressed by a specific shRNA or transfected with an overexpression plasmid in the SH-SY5Y cells. Under the exposure to rotenone or not, the expression of constitutive proteasome β subunits, i-proteasome βi subunits, antigen presentation related proteins, α-syn and TH were detected by Western blot in PSMB9-silenced or -overexpressed cells, and the proteasomal activities were detected by fluorogenic peptide substrates. The location of i-proteasome βi subunits and α-syn were detected by immunofluorescence staining. The levels of ROS, GSH and MDA were measured by commercial kits. Cell apoptosis was detected by flow cytometry. Besides impairing the constitutive proteasomes, rotenone induced the expression of βi subunits of i-proteasome and antigen presentation related proteins such as TAP1, TAP2 and MHC-I. Silencing or overexpressing PSMB9 had no obvious effect on the levels of other subunits, but could regulate the chymotrypsin-like activity of 20S proteasome and the expression of TAP1, TAP2 and MHC-I. Three βi subunits (PSMB9, PSMB10, PSMB8) of i-proteasome were all co-localized with α-syn. PSMB9 knockdown aggravated accumulation of α-syn, degradation of TH, release of ROS, increased level of MDA, decreased level of GSH and eventually promoted apoptosis in SH-SY5Y cells after rotenone treatment, while over-expression of PSMB9 could attenuate these toxic effects of rotenone. I-proteasome is activated in SH-SY5Y cells treated with rotenone and may play a neuroprotective role.

Background and purpose In addition to the central nervous system-mediated action, leptin also directly induces fatty acid oxidation in skeletal muscle. Rapid induction of FAO by leptin is mediated by the AMP-activated protein kinase (AMPK) pathway, but the mechanism of prolonged FAO by leptin was previously unknown. In an earlier study, we showed that free fatty acids increase transcription of small ubiquitin-like modifier (SUMO) specific protease 2 (SENP2) in skeletal muscle, and that SENP2 stimulates expression of FAO-associated enzymes by deSUMOylating peroxisome proliferator-activated receptors, PPARδ and PPARγ. In this study, we examine whether SENP2 is involved in prolonged stimulation of FAO by leptin. Methods The Effect of leptin on expression of SENP2 and on SENP2-mediated FAO was investigated by using western blotting and real time qPCR of C2C12 myotubes, and of C2C12 myotubes in which expression of specific genes was knocked down using siRNAs. Additionally, muscle-specific SENP2 knockout mice were generated to test the involvement of SENP2 in leptin-induced FAO in vivo. Results We show that leptin treatment of C2C12 myotubes causes signal transducer and activator of transcription 3 (STAT3) to bind to the Senp2 promoter, inducing SENP2 expression. We also show that leptin increases the binding of PPARδ and PPARγ to PPRE sites in the promoters of two FAO-associated genes: long-chain acyl-CoA synthetase 1 (Acsl1) or carnitine palmitoyl transferase 1b (Cpt1b). When SENP2 is knocked down in myotubes, leptin-induced expression of FAO-associated enzymes and prolonged increase of FAO are suppressed, but rapid increase of FAO is unaffected. In addition, leptin-induced expression of FAO-associated enzymes was not observed in muscle tissue of SENP2 knockout mice. Conclusions We demonstrate that the peripheral actions of leptin on FAO are mediated by two different pathways: AMPK causes a rapid increase in FAO, and SENP2 of the STAT3 pathway causes a slow, prolonged increase in FAO.

Legumain is a proteolytic enzyme that plays a role in the regulation of cell proliferation in invasive breast cancer. Studies evaluating its role in ductal carcinoma in situ (DCIS) are lacking. Here, we aimed to characterize legumain protein expression in DCIS and evaluate its prognostic significance. Legumain was assessed immunohistochemically in a tissue microarray of a well-characterized cohort of DCIS (n = 776 pure DCIS and n = 239 DCIS associated with invasive breast cancer (DCIS-mixed)). Legumain immunoreactivity was scored in tumor cells and surrounding stroma and related to clinicopathological parameters and patient outcome. High legumain expression was observed in 23% of pure DCIS and was associated with features of high-risk DCIS including higher nuclear grade, comedo necrosis, hormone receptor negativity, HER2 positivity, and higher proliferation index. Legumain expression was higher in DCIS associated with invasive breast cancer than in pure DCIS (p

Hand-foot-mouth disease (HFMD) is a common viral disease in young children, mainly caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Specific antiviral agents are not commercially available yet. Here we report that the macrolide antibiotics spiramycin (SPM) and azithromycin (AZM) possess antiviral activities against EV-A71 and CV-A16. SPM significantly reduced EV-A71 RNA and protein levels, most likely through interfering with viral RNA replication. The SPM-resistant EV-A71 variants showed similar resistance to AZM, indicating a similar anti-EV-A71 mechanism by which these two drugs exert their functions. The mutations of these variants were reproducibly mapped to VP1 and 2A, which were confirmed to confer resistance to SPM. Animal experiments showed that AZM possesses stronger anti-infection efficacy than SPM, greatly alleviated the disease symptoms and increased the survival rate in a mouse model severely infected with EV-A71. In all, our work suggests that AZM is a potential treatment option for EV-A71-induced HFMD, whose proved safety for infants and children makes it even more promising.

Autophagy plays an important role in the fight against Mycobacterium tuberculosis infection. Massive researches proved that miRNAs could be the regulators of autophagy, which implied miRNAs could favor MTB invasion or latent infection. In our study, multiple bioinformatics databases and software were used to seek and lock the miRNAs associating with regulation of autophagy. Notably, a novel miR-129-3p was found and its target gene Atg4b showed grand potential in mediation of autophagy. Moreover, BCG infection triggered miR-129-3p overexpression in RAW264.7 cells. Up-regulation of miR-129-3p decreased mRNA or protein level of Atg4b and resulted in the inhibition of autophagy. The antagomir of miR-129-3p had the opposite impact. The LC3 puncta formation in RAW264.7 cells were also affected after transfection of miR-129-3p mimic or antagomir. The mRFP-GFP-LC3 analysis indicated that mimic of miR-129-3p impaired autophagic flux while antagomir improved autophagy. The CFU assay results showed that miR-129-3p promoted the intracellular survival of BCG in macrophages. Consequently, these data suggested that miR-129-3p could favor MTB survival by inhibiting autophagy via Atg4b.

Conjugation of avian IgY antibodies to nanosensors has been extensively explored for the diagnostics of virus and parasite infection, as well as for the detection of pharmaceutically and toxicologically relevant molecules. However, to date this strategy has only been minimally applied the detection of allergens. In this study, gold nanoparticles (GNPs) were conjugated to a polyvalent IgY antibodies raised against Dermatophagoides group I allergens. GNPs were synthesized by HAuCL4 reduction using 1% trisodium citrate, and characterized them by absorption spectroscopy and transmission electron microscopy (TEM). The most stable immunoconjugates were obtained with 18-nm monodisperse GNPs and a minimal concentration of 12.5 μg/mL of IgY at pH 7.5. The immunoconjugate was capable of detecting up to 1.5 μg of a total Dermatophagoides farinae protein extract in an immuno-dot blot assay. This immunoreactant conjugate represents a new tool for the detection and control of indoor dust mite allergens.

Cysteine cathepsins have been for a long time considered to execute mainly nonspecific bulk proteolysis in the endolysosomal system. However, this view has been changing profoundly over the last decade as cathepsins were found in the cytoplasm, nucleus and in the extracellular milieu. Cathepsins are currently gaining increased attention largely because of their extracellular roles associated with disease development and progression. While kept under tight control under physiological conditions, their dysregulated and elevated activity in the extracellular milieu are distinctive hallmarks of numerous diseases such as various cancers, inflammatory disorders, rheumatoid arthritis, bone disorders and heart diseases. In this review, we discuss cysteine cathepsins with a major focus on their extracellular roles and extracellular proteolytic targets beyond degradation of the extracellular matrix. We further highlight the perspectives of cathepsin research and novel avenues in cathepsin-based diagnostic and therapeutic applications.

Graphical abstract, A non-prime site substituent and warheads combined with a decahydroisoquinolin scaffold was evaluated as a novel inhibitor for severe acute respiratory syndrome (SARS) chymotrypsin-like protease (3CLpro). The decahydroisoquinolin scaffold has been demonstrated to be an effective hydrophobic center to interact with S2 site of SARS 3CLpro, but the lack of interactions at S3 to S4 site is thought to be a major reason for the moderate inhibitory activity. In this study, the effects of an additional non-prime site substituent on the scaffold as well as effects of several warheads are evaluated. For the introduction of a desired non-prime site substituent, amino functionality was introduced on the decahydroisoquinolin scaffold, and the scaffold was constructed by Pd(II) catalyzed diastereoselective ring formation. The synthesized decahydroisoquinolin inhibitors showed about 2.4 times potent inhibitory activities for SARS 3CLpro when combined with a non-prime site substituent. The present results indicated not only the expected additional interactions with the SARS 3CLpro but also the possibility of new inhibitors containing a fused-ring system as a hydrophobic scaffold and a new warhead such as thioacetal.