Serine-type Protease Research Articles

The serine peptidase inhibitor N-ρ-tosyl-l-phenylalanine chloromethyl ketone (TPCK) affects the cell biology of Candida haemulonii species complex

Candida haemulonii species complex (C. haemulonii, C. haemulonii var. vulnera and Candida duobushaemulonii) is composed by emerging and multidrug-resistant (MDR) yeasts. Candidiasis, the disease caused by these species, is difficult to treat and culminates in clinical failures and patient death. It is well-known that Candida peptidases play important roles in the fungus–host interactions, and hence these enzymes are promising targets for developing new antifungal drugs. Recently, serine-type peptidases were described in clinical isolates of C. haemulonii complex with the ability to cleave relevant key host proteins. Herein, the effects of serine peptidase inhibitors (SPIs) on the cell biology of this fungal complex were evaluated. Initially, eight distinct SPIs (phenylmethylsulfonyl fluoride – PMSF, 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride – AEBSF, N-α-tosyl-l-lysine chloromethyl ketone hydrochloride – TLCK, N-p-tosyl-l-phenylalanine chloromethyl ketone – TPCK, simeprevir, boceprevir, danoprevir and telaprevir) were tested on the fungal growth. TPCK showed the best efficacy in controlling cell proliferation, being selected for the following experiments. This SPI induced changes in the architecture of yeast cells, as observed by scanning electron microscopy, besides injuries at the plasma membrane and reduction in the ergosterol content. TPCK also diminished the ability of yeasts to adhere to abiotic (polystyrene and glass) and biotic (murine macrophages) surfaces in a typically concentration-dependent manner. In addition, the 24 h-treatment of the mature biofilm promoted a decrease in biomass, viability and extracellular matrix. Altogether, our results highlight that SPIs may be promising new therapeutic agents in the treatment of candidiasis caused by emergent, opportunistic and MDR species forming the C. haemulonii complex.

Cloning, expression and characterization of P1 and NIa proteases from banana bract mosaic virus (BBrMV)

Banana bract mosaic virus (BBrMV) causes the banana bract mosaic disease in banana. It belongs to the genus Potyvirus within the family Potyviridae. To the best of our knowledge apart from BBrMV coat protein gene, there are no reports on cloning, expression and characterization of any other genes from BBrMV. In this study, the BBrMV P1 and NIa protease genes were amplified from BBrMV infected banana plant cultivar Nendran and were cloned into the protein expression vector pET28b. Recombinant plasmids were transferred to BL21-CodonPlus (DE3)-RP cells and the IPTG (Isopropyl β-d-1-thiogalactopyranoside) induced BBrMV P1 and NIa proteins with molecular weights of 42 and 32 KDa respectively were purified on Ni-NTA resin column under denaturing conditions using 8 M urea. BBrMV P1 and NIa purified proteins were detected by Western blot using anti-histidine antibody. The activity of both P1 and NIa proteases in native form was analyzed through in-gel zymographic assay. The activities of both the proteases were strongly inhibited by PMSF, suggesting that both the proteases are the serine type proteases. Interestingly both the proteases showed a temperature optimum of 50 °C while the pH optimum was 8. Both proteases lost their activity when incubated at 70 °C for 1 h. This is the first report of expression, purification and characterization of BBrMV P1 and NIa proteases.

Tripeptidyl peptidase I promotes human endometrial epithelial cell adhesive capacity implying a role in receptivity

The endometrium undergoes cyclic remodelling throughout the menstrual cycle in preparation for embryo implantation which occurs in a short window during the mid-secretory phase. It is during this short ‘receptive window’ that the endometrial luminal epithelium acquires adhesive capacity permitting blastocysts firm adhesion to the endometrium to establish pregnancy. Dysregulation in any of these steps can compromise embryo implantation resulting in implantation failure and infertility. Many factors contribute to these processes including TGF-β, LIF, IL-11 and proteases. Tripeptidyl peptidase 1 (TPP1) is a is a lysosomal serine-type protease however the contribution of the TPP1 to receptivity is unknown. We aimed to investigate the role of TPP1 in receptivity in humans.In the current study, TPP1 was expressed in both epithelial and stromal compartments of the endometrium across the menstrual cycle. Expression was confined to the cytoplasm of luminal and glandular epithelial cells and stromal cells. Staining of mid-secretory endometrial tissues of women with normal fertility and primary unexplained infertility showed reduced immunostaining intensity of TPP1 in luminal epithelial cells of infertile tissues compared to fertile tissues. By contrast, TPP1 levels in glandular epithelial and stromal cells were comparable in both groups in the mid-secretory phase. Inhibition of TPP1 using siRNA compromised HTR8/SVneo (trophoblast cell line) spheroid adhesion on siRNA-transfected Ishikawa cells (endometrial epithelial cell line) in vitro. This impairment was associated with decreased sirtuin 1 (SIRT1), BCL2 and p53 mRNA and unaltered, CD44, CDH1, CDH2, ITGB3, VEGF A, OSTEOPONTIN, MDM2, CASP4, MCL1, MMP2, ARF6, SGK1, HOXA-10, LIF, and LIF receptor gene expression between treatment groups. siRNA knockdown of TPP1 in primary human endometrial stromal cells did not affect decidualization nor the expression of decidualization markers prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1). Taken together, our data strongly suggests a role for TPP1 in endometrial receptivity via its effects on epithelial cell adhesion and suggests reduced levels associated with unexplained infertility may contribute to implantation failure.

GASPIDs Versus Non-GASPIDs - Differentiation Based on Machine Learning Approach

Background: Peptidases are a group of enzymes which catalyze the cleavage of peptide bonds. Around 2-3% of the whole genome codes for proteases and about one-third of all known proteases are serine proteases which are divided into 13 clans and 40 families. They are involved in diverse physiological roles such as digestion, coagulation of blood, fibrinolysis, processing of proteins and prohormones, signaling pathways, complement fixation, and have a vital role in the immune defense system. Based on their functions, they can broadly be divided into two classes; GASPIDs (Granule Associated Serine Peptidases involved in Immune Defense System) and Non- GASPIDs. GASPIDs, in particular are involved in immune-associated functions i.e. initiating apoptosis to kill virally infected and cancerous cells, cytokine modulation for the generation of inflammatory responses, and direct killing of pathogens through phagosomes. Methods: In this study, sequence-based characterization of these two types of serine proteases is performed. We first identified sequences by analyzing multiple online databases as well as by analyzing whole genomes of different species from different orthologous and non-orthologous species. Sequences were identified by devising a distinct criterion to differentiate GASPIDs from Non-GASPIDs. The translated version of these sequences was then subjected to feature extraction. Using these distinctive features, we differentiated GASPIDs from Non-GASPIDs by applying multiple supervised machine learning models. Results and Conclusion: Our results show that, among the three classifiers used in this study, SVM classifier coupled with tripeptide as feature method has shown the best accuracy in classification of sequences as GASPIDs and Non-GASPIDs.

Abstract 2212: An antibody drug conjugate targeting activated matriptase, exhibits synergistic cytotoxicity with a PARP inhibitor, olaparib in triple negative breast cancer

Abstract Matriptase, is a serine protease type II transmembrane protein. Matriptase forms a cognate pair with its Kunitz type endogenous inhibitor HAI-1 and is commonly deregulated and ubiquitously expressed in a variety of human carcinomas and in particular, breast cancer. This enzyme activity is regulated by binding to its endogenous inhibitors (HAI-1 or 2). Previously, we reported the synthesis and anti-tumor studies of a novel antibody drug conjugate (ADC) targeting activated matriptase of tumor cells (Oncotarget, 2018,9(40):25983). The activated matriptase-specific monoclonal antibody (M69) was conjugated with a potent anti-mitotic toxin, monomethyl auristatin-E (MMAE) via a lysosomal protease (cathepsin B) -cleavable dipeptide linker. The ADC was found to be potent and selective against multiple activated matriptase-positive epithelial carcinoma cell lines including triple negative breast cancer (TNBC), as well as alone and in combination with bortezomib in Mantle cell lymphoma (Frontiers in Oncology, 2019,9:258). In-vivo studies showed that the ADC has potent anti-tumor effects alone or in combination with cisplatin or carboplatin against TNBC xenografts and a primary human TNBC (PDX), respectively. The chimeric ADC (mouse-human chimera) also evoked an immune response against TNBC xenografts in humanized mouse models. Here, we report the sensitivity of TNBC tumors with and without a BRCA mutation to ADC alone or in combination with a PARP inhibitor, Olaparib. We analyzed the mutational status of BRCA1, BRAC2, p53 and activated matriptase expression of four TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC1806 and MDA-MB-436). All of the cell lines had p53 mutations. The MDA-MB-468 cell line with a mutation in BRCA 2, and expression of activated matriptase was the most sensitive cell line to both olaparib and the chimeric MMAE ADC, and the combination of them resulted in marked synergistic cell kill. The MDA-MB-436 cell line with a splice site mutation in BCRA1 was less sensitive to both treatments, but there was modest synergy when the two drugs were combined. In contrast the MDA-MB-231 cell line without a BRCA mutation was much less sensitive to the ADC or olaparib, and the combination did not show synergy. The HCC1806 cell line with wt BRCA was sensitive to the ADC and olaparib, but the combination resulted in antagonistic effects. Triple negative breast cancers with a BRAC2 mutation and overexpression of activated matriptase may be a subset of breast cancers that may benefit from the combination of our novel ADC and a PARP inhibitor. Citation Format: Gulam M. Rather, Zoltan Szekely, Joseph R. Bertino. An antibody drug conjugate targeting activated matriptase, exhibits synergistic cytotoxicity with a PARP inhibitor, olaparib in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2212.

Probing contacts of inhibitor locked in transition states in the catalytic triad of DENV2 type serine protease and its mutants by 1H, 19F and 15\u2009N NMR spectroscopy

BackgroundDetailed structural knowledge of enzyme-inhibitor complexes trapped in intermediate state is the key for a fundamental understanding of reaction mechanisms taking place in enzymes and is indispensable as a structure-guided drug design tool. Solution state NMR uniquely allows the study of active sites of enzymes in equilibrium between different tautomeric forms. In this study 1H, 19F and 15 N NMR spectroscopy has been used to probe the interaction contacts of inhibitors locked in transition states of the catalytic triad of a serine protease. It was demonstrated on the serotype II Dengue virus NS2B:NS3pro serine protease and its mutants, H51N and S135A, in complex with high-affinity ligands containing trifluoromethyl ketone (tfk) and boronic groups in the C-terminal of tetra-peptides.ResultsMonitoring 19F resonances, shows that only one of the two isomers of the tfk tetra-peptide binds with NS2B:NS3pro and that access to the bulk of the active site is limited. Moreover, there were no bound water found in proximity of the active site for any of the ligands manifesting in a favorable condition for formation of low barrier hydrogen bonds (LBHB) in the catalytic triad. Based on this data we were able to identify a locked conformation of the protein active site. The data also indicates that the different parts of the binding site most likely act independently of each other.ConclusionsOur reported findings increases the knowledge of the detailed function of the catalytic triad in serine proteases and could facilitate the development of rational structure based inhibitors that can selectively target the NS3 protease of Dengue type II (DENV2) virus. In addition the results shows the usefulness of probing active sites using 19F NMR spectroscopy.

The transcription factor PrtT and its target protease profiles in Aspergillus niger are negatively regulated by carbon sources.

To survey genome-scale protease profiles regulated by the Aspergillus niger transcription factor PrtT and further controlled by carbon sources. The PrtT disruption mutant (delprtT) and overexpression (OEprtT) strains were successfully generated and further confirmed by phenotypic and protease activity analysis. RNA-seq analysis of WT and mutants identified 32 differentially expressed protease genes, which mostly belonged to serine-type peptidases, aspartic-type endopeptidases, aminopeptidases and carboxypeptidases. Furthermore, based on the MEME predicted motif analysis of the PrtT promoter, EMSA and phenotypic and qRT-PCR analyses confirmed that the carbon metabolism regulator AmyR directly regulated the protease genes and their regulatory factor PrtT. Thirty-two PrtT-regulated protease genes were identified by RNA-seq, and the secondary carbon source regulator AmyR was found to have a negative regulatory effect on the expression of PrtT and its target protease genes.

Bioprospecting Psychrotrophic Bacteria for Serine-Type Proteases from the Cold Areas of Western Himalayas.

The aim of present study was to analyze the prevalence of protease diversity among psychrotrophic bacteria in Lahaul and Spiti of the Western Himalayas. A total of 459 bacteria were screened and protease activity was observed in 150 isolates at 5°C. Furthermore, 55 isolates showed protease activity up to pH 10 at 5°C. Based on the hydrolytic zone, 22 isolates were selected for protease quantification. The protease activity varied from 58-377UmL-1 at 10°C, 49-396UmL-1 at 28°C and 31-407UmL-1 at 37°C. Similarly, protease activity ranged from 36-353UmL-1 at pH 7, 40-306UmL-1 at pH 9 and 33-304UmL-1 at pH 10. The isolates were identified based on 16S rRNA gene sequencing and showed phylogenetic relationship to Arthrobacter belonging to the class Actinobacteria, Bacillus, Exiguobacterium, Paenibacillus, and Planomicrobium to Bacilli, and Pseudomonas, Serratia, and Stenotrophomonas to Gammaproteobacteria. Zymogram analysis revealed variations in protease isoforms ranging from 20 to 250kDa which were strongly inhibited in the presence of phenylmethylsulfonyl fluoride, thus indicated serine-type nature. The extensive number of serine proteases among these bacteria was confirmed by annotating genomes of the reported genera for prevalence of protease isoforms. The properties of proteases including low-temperature activity with alkaline stability and detergent compatibility suggested their suitability as bio-additives in laundry.

Extracellular protease profile of Acanthamoeba after prolonged axenic culture and after interaction with MDCK cells.

Free-living amoebae of the genus Acanthamoeba are causative agents of Acanthamoeba keratitis and amoebic encephalitis in humans, both of which are serious infections. The ability to produce proteases is one of the factors involved in the pathogenesis of Acanthamoeba infections. The aim of this study was to evaluate the secreted proteases of six Acanthamoeba strains from distinct genotypes (T1, T2, T4 and T11) maintained in prolonged axenic culture and following three successive passages in Madin-Darby Canine Kidney (MDCK) cells. Conditioned medium was obtained from cultures before and after interaction with the MDCK monolayers, resolved in SDS-PAGE containing gelatine, then subjected to quantitative azocasein assays. Zymography profiles varied between the strains, with the predominant proteases found to be serine-type proteases from 49 to 128 kDa. A T1 genotype strain isolated from dust showed quantitatively higher protease secretion compared to the other strains. No changes were detected in the zymography profiles of MDCK-interacted cultures compared to long-term axenic cultures. Two strains presented lower proteolytic activity post-MDCK interaction, while the remaining strains presented similar values before and after MDCK passages. In conclusion, this study confirms the predominance of serine-type protease secretion by Acanthamoeba, with distinct profiles presented by the different strains and genotypes studied. Also, interaction of trophozoites with MDCK cells did not alter the zymography pattern.

Chihuil Sea Catfish Bagre panamensis Viscera as a New Source of Serine Proteases: Semi-purification, Biochemical Characterization and Application for Protein Hydrolysates Production

The recovery of proteases from fish viscera could be a strategy to reduce environmental problems caused by inadequate disposal of fish by-products. This study reports the biochemical characterization of proteases isolated from chihuil sea catfish (Bagre panamensis) intestines and the evaluation of their stability to different physical and chemical factors. Protein hydrolysates from chihuil muscle and casein were produced using its semi-purified proteases extract (SPE) and alcalase. Assays with specific protease inhibitors indicate that trypsin and chymotrypsin are the main types of serine proteases in SPE. Semi-purified enzymes exhibited proteolytic activity at alkaline pH (9–12), and high stability at low/mild temperatures (10–40 °C). A 92% of SPE proteolytic activity was retained in the presence of 30% NaCl. The enzyme extract was stable in reducing agents (2-mercaptoethanol and DTT) but lost about 70% of proteolytic activity in anionic detergents like SDS and tween-80. Organic solvents did not affect the enzyme activity of SPE. Finally, maintaining a same E/S ratio for protein hydrolysates elaboration, chihuil serin proteases exhibited a higher hydrolytic efficiency compared to alcalase when casein and proteins from chihuil muscle were hydrolyzed. Thus, the semi-purification of serine proteases from chihuil viscera provided a low-cost source of enzymes with interesting catalytic features.

Gluten-Degrading Proteases in Wheat Infected by Fusarium graminearum-Protease Identification and Effects on Gluten and Dough Properties.

Recently, we have observed a relationship between poor breadmaking quality and protease activities related to fungal infection. This study aims to identify potential gluten-degrading proteases secreted by fungi and to analyze effects of these proteases on rheological properties of dough and gluten. Fusarium graminearum-infected grain was used as a model system. Zymography showed that serine-type proteases secreted by F. graminearum degrade gluten proteins. Zymography followed by liquid chromatography-mass spectrometry (MS)/MS analysis predicted one serine carboxypeptidase and seven serine endo-peptidases to be candidate fungal proteases involved in gluten degradation. Effects of fungal proteases on the time-dependent rheological properties of dough and gluten were analyzed by small amplitude oscillatory shear rheology and large deformation extensional rheology. Our results indicate that fungal proteases degrade gluten proteins not only in the grain itself, but also during dough preparation and resting. Our study gives new insights into fungal proteases and their potential role in weakening of gluten.

Hemostatically potent small molecular weight serine protease from Maclura spinosa (Roxb. ex Willd.) accelerates healing of subcutaneous dermal wounds in Swiss albino mice

Latex of Maclura spinosa has been used by Kollamalayali tribal community of Western Ghats for curdling of milk. In our previous study we have reported the presence of multiple proteases in the latex, among which the serine protease possesses potent role in hemostasis. With a view to further characterize these proteases the current study was taken up. Maclura spinosa latex contains a serine protease which is resolved using molecular size exclusion column chromatography and an anion exchanger resin in a consecutive manner. The specific activity of the enzyme Maclura spinosa latex protease (MSLP) was found to be 56.15 units/mg and recovery to be 2.68% with a fold purity of 0.41. Being a typical serine protease, MSLP is significantly inhibited by PMSF up to 72.97%. The optimum temperature and pH for the enzyme were found to be 50 °C and 8 respectively. Excision wound healing assay in Swiss albino mice using MSLP, showed accelerated wound closure up to 89.35 ± 1.209% compared to 91.938 ± 1.649% shown by positive control. Further PMSF treated MSLP sample did not show considerable wound healing which confirms exclusive involvement of serine proteases. Examination of biochemical markers viz, hydroxy proline content in healing tissue and catalase activity of fatty tissue also ascertain the potential of MSLP in wound healing. Histopathological studies of healing tissue provide confirmatory evidence with dense collagenation of tissue and fibroblast proliferation rendered by MSLP in the respective treated subjects.

Abstract 550: Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity

While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naive donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities. Citation Format: Steven C. Pai, Donald M. Simons, Xiaoqing Lu, Wendy Ritacco, Michael Evans, Gillian Kingsbury, Lawrence Fong. Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 550.

Abstract 550: Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity

Abstract While immune checkpoint blockade leads to potent anti-tumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in multi-organ inflammation including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA-4 antibody (anti-CTLA-4 DVD) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA-4 binding domain. Upon reaching the tumor, the outer domain is cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA-4 blockade. Anti-CTLA-4 DVD significantly reduced multi-organ immune toxicity by preserving tissue-resident regulatory T cells (Tregs) in Rag 1-/- mice that received naïve donor CD4+ T cells from wild-type C57BL/6j mice. Moreover, anti-CTLA-4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2 bearing C57BL/6j mice. Tregs depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA-4 without the FcγR binding portion (anti-CTLA-4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrated a new approach to anti-CTLA-4 blockade that depletes tumor-infiltrating, but not tissue-resident Tregs, preserving anti-tumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA-4 DVD provides an avenue to uncouple anti-tumor efficacy from immunotherapy-induced toxicities. Citation Format: Steven C. Pai, Donald M. Simons, Xiaoqing Lu, Wendy Ritacco, Michael Evans, Gillian Kingsbury, Lawrence Fong. Tumor-conditional anti-CTLA-4 uncouples anti-tumor efficacy from immunotherapy-related toxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 550.

Alzheimer's Amyloidopathy: An Alternative Aspect.

The ‘amyloid hypothesis’ dominates Alzheimer’s disease (AD) research but has failed to deliver effective therapies. Amyloid precursor protein (APP) and presenilin-1 (PSEN1) genetic mutations are undoubtedly pathogenic, albeit by unclear mechanisms. Conversely, high dose B-vitamins convincingly slow brain atrophy in a pre-stage state of sporadic AD. Here we suggest a link between sporadic and genetic AD: 1) Increased serum homocysteine, a marker of B-vitamin deficiencies, is a significant risk factor for sporadic AD. It also correlates with elevated levels of antichymotrypsin, a serine protease inhibitor. 2) Family members with codon 717 APP mutations and dementia have low serum vitamin B12 values. Overexpression of the APP domain coding for a Kunitz type serine protease inhibitor might explain this. 3) PSEN1 mutations disrupt lysosomal function due to reduced proteolytic activity. They also trap cobalamin (B12) within lysosomes, leading to intracellular deficiency of the vitamin. In summary, APP and PSEN1 mutations both confer a risk for reduced protease activity and B12 bio-availability. Comparably, sporadic AD features a constellation of increased protease inhibition and B-vitamin deficiencies, the central part of which is believed to be B12. These concordant observations in three disparate AD etiologies suggest a common neuropathogenic pathway. This hypothesis is evaluable in laboratory and clinical trials.

Identification of key pathways and candidate genes in pancreatic ductal adenocarcinoma using bioinformatics analysis.

Pancreatic ductal adenocarcinoma (PDAC) is a malignant tumor with a high degree of malignancy that is difficult to diagnose and treat. The present study integrated PDAC cohort profile datasets to identify key candidate genes and pathways involved in the pathogenesis of the disease. The expression profiles of GSE28735 included 45 PDCA and matching pairs of adjacent non-tumor tissue. Differentially expressed genes (DEGs) were sorted and candidate genes and pathway enrichment were analyzed. A DEG-associated protein-protein interaction (PPI) network was constructed. A total of 424 DEGs were identified in PDAC, including 159 upregulated genes and 265 downregulated genes. Gene Ontology analysis results indicated that upregulated DEGs were significantly enriched in biological process, molecular function and cellular component categories. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that the upregulated DEGs were enriched in ‘pancreatic secretion’, ‘protein digestion’ and ‘absorption’. Downregulated DEGs were enriched in ‘ECM-receptor interaction’, ‘focal adhesion’ and ‘PI3K/AKT’ signaling pathways. The PPI network revealed that these genes were involved in significant pathways, including ‘ECM organization’ signaling pathways (Hippo signaling pathway, TGF-β signaling pathway, Hedgehog signaling pathway and Wnt signaling pathway), ‘serine-type peptidase activity’ signaling pathway (PI3K-Akt signaling pathway, TNF-α signaling pathway and Wnt signaling pathway) and ‘extracellular region’ signaling pathways (RTP signaling pathway, G protein-coupled receptor signaling pathway and RAS-RAF-MAPK signaling pathway). The identification of these candidate genes and pathways sheds light on the etiology and molecular mechanisms of PDAC and may guide the development of novel therapies for pancreatic cancer.

Proteomics study of Southern Punjab Pakistani cobra (Naja naja: formerly Naja naja karachiensis) venom

Because of the potential of significant geographic variation in the compositions of snake venoms, much insight can be gained by examining individuals originating from diverse locations, and detailed characterization of any species can only be done by including animals from the entire range. Pakistani N. naja (formerly N. naja karachiensis) venom was decomplexed by applying diverse techniques and found a concoction of proteins (a total of 43 venomous proteins comprising 11 super families) from 6 to 200 kilodaltons in size. Among them three finger toxins (58%), phosphoplipses A2 (19%), snake venom metalloproteinases (5%), l-amino acid oxidases (5%), helvepryns (3%), vespryns (2%), cobra venom factor (2%), 5′-nucleotidases (2%), venom nerve growth factor (2%), and Kunitz type serine protease inhibitor (2%) were included. It is the first report that this venom possesses significantly higher percentage of three finger toxins belonging to the cytotoxins (32% overall) and α‐neurotoxins (32% overall, predominately long chain neurotoxins, 24% overall) compared to N. naja from other geographic locations. Better understanding of intraspecific variations in venom can improve the development of location-specific anti-venoms. Furthermore, this pool of diverse toxins could potentially be a source of novel drug candidates for the treatment of muscarinic acetylcholine receptors mediated nervous disorders.

Immunonutritional consequences of different serine-type protease inhibitors in a C57BL/6 hepatocarcinoma model.

Imbalances in innate immunity and the activity of innate immune cells are implicated in the development of hepatocellular carcinoma (HCC). Plant seeds are good sources of protease inhibitors, which can have a significant influence on human health disorders, especially in the field of cancer prevention. To elucidate the impact and preventive effects of immunonutritional serine-type protease inhibitors (STPIs) on HCC, it was used an established model of chemically induced liver injury. Injured livers induced Akt as well as hepatic infiltration of NKG2D+ and CD74+ cells. Feeding STPIs reduced size and number of intrahepatic nodes of mononuclear. These animals showed an inverse association of the severity of HCC with bioactive hepcidin levels, which was significantly correlated with the hepatic myeloperoxidase activity. According to their origin, administration of STPIs significantly induce increased numbers of F4/80+ cells in injured livers that can be responsible for the biological effects detected on the parenchyma and inflammatory markers under DEN/TAA treatment. These findings can have direct implications in HCC immunotherapy where enhanced response(s) in inflammation-driven cancer patients could help promoting inflammation-driven processes and favor tumor growth. Altogether, this study demonstrates that oral administration of STPIs modulate innate immunity response influencing HCC aggressiveness and progression. These results represent a path forward to develop durable, long-lasting response against hepatocarcinoma and open a future research path in the development of coadjutant intervention strategies to pharmacological therapies.

Plant seed protease inhibitors differentially affect innate immunity in a tumor microenvironment to control hepatocarcinoma.

Identifying tolerance responses to ingested foodstuff over life is essential for understanding dysfunction in metabolic diseases. This study presents a comparative structural and functional analysis of serine-type protease inhibitors (STPIs) from Chenopodium quinoa, Salvia hispanica L., Avena sativa and Triticum durum. It also evaluates their influence on an in vivo hepatocarcinoma (HCC) model. STPIs are found in all samples with significant differences in protease inhibitory capacity: C. quinoa = S. hispanica < A. sativa = T. durum. STPIs in C. quinoa and S. hispanica appear as heterologous complexes, while those in A. sativa are present as homologous complexes. T. durum provides different subunits with STPI capacity. HPLC-RP-ESI analyses revealed homology between STPIs in the different samples and the partial resistance of those to simulated gastrointestinal digestion. In vivo, STPIs from S. hispanica showed the most positive effects, increasing F4/80+ cells normalizing the expression (mRNA) of CD36 and the innate immune 'Toll-like' receptor (TLR)-4. Only STPIs from C. quinoa and S. hispanica did not impair the production of inflammatory mediators (granulocyte-monocyte colony stimulating factor, stem cell factor and TNFα), contributing to maintaining the polarization of the antitumoral M1 macrophage phenotype. These structural and functional features of STPIs from C. quinoa and S. hispanica can be used to control HCC aggressiveness.

Tumor-conditional anti-CTLA4 uncouples antitumor efficacy from immunotherapy-related toxicity.

While immune checkpoint blockade leads to potent antitumor efficacy, it also leads to immune-related adverse events in cancer patients. These toxicities stem from systemic immune activation resulting in inflammation of multiple organs, including the gastrointestinal tract, lung, and endocrine organs. We developed a dual variable domain immunoglobulin of anti-CTLA4 antibody (anti-CTLA4 DVD, where CTLA4 is defined as cytotoxic T lymphocyte-associated antigen-4) possessing an outer tumor-specific antigen-binding site engineered to shield the inner anti-CTLA4-binding domain. Upon reaching the tumor, the outer domain was cleaved by membrane type-serine protease 1 (MT-SP1) present in the tumor microenvironment, leading to enhanced localization of CTLA4 blockade. Anti-CTLA4 DVD markedly reduced multiorgan immune toxicity by preserving tissue-resident Tregs in Rag 1-/- mice that received naive donor CD4+ T cells from WT C57BL/6j mice. Moreover, anti-CTLA4 DVD induced potent antitumor effects by decreasing tumor-infiltrating Tregs and increasing the infiltration of antigen-specific CD8+ T lymphocytes in TRAMP-C2-bearing C57BL/6j mice. Treg depletion was mediated through the antibody-dependent cellular cytotoxicity (ADCC) mechanism, as anti-CTLA4 without the FcγR-binding portion (anti-CTLA4 DANA) spared Tregs, preventing treatment-induced toxicities. In summary, our results demonstrate an approach to anti-CTLA4 blockade that depletes tumor-infiltrating, but not tissue-resident, Tregs, preserving antitumor effects while minimizing toxicity. Thus, our tumor-conditional anti-CTLA4 DVD provides an avenue for uncoupling antitumor efficacy from immunotherapy-induced toxicities.