Calpain-2 participates in the process of calpain-1 inactivation

Abstract Calpain-1 and calpain-2 are highly structurally similar isoforms of calpain. The calpains, a family of intracellular cysteine proteases, cleave their substrates at specific sites, thus modifying their properties such as function or activity. These isoforms have long been considered to function in a redundant or complementary manner, as they are both ubiquitously expressed and activated in a Ca2+- dependent manner. However, studies using isoform-specific knockout and knockdown strategies revealed that each calpain species carries out specific functions in vivo. To understand the mechanisms that differentiate calpain-1 and calpain-2, we focused on the efficiency and longevity of each calpain species after activation. Using an in vitro proteolysis assay of troponin T in combination with mass spectrometry, we revealed distinctive aspects of each isoform. Proteolysis mediated by calpain-1 was more sustained, lasting as long as several hours, whereas proteolysis mediated by calpain-2 was quickly blunted. Calpain-1 and calpain-2 also differed from each other in their patterns of autolysis. Calpain-2–specific autolysis sites in its PC1 domain are not cleaved by calpain-1, but calpain-2 cuts calpain-1 at the corresponding position. Moreover, at least in vitro, calpain-1 and calpain-2 do not perform substrate proteolysis in a synergistic manner. On the contrary, calpain-1 activity is suppressed in the presence of calpain-2, possibly because it is cleaved by the latter protein. These results suggest that calpain-2 functions as a down-regulation of calpain-1, a mechanism that may be applicable to other calpain species as well.

Download Full-text

Siderophore-mediated zinc acquisition enhances enterobacterial colonization of the inflamed gut

10.1101/2020.07.20.212498 ◽

2020 ◽

Author(s):

Hui Zhi ◽

Judith Behnsen ◽

Allegra Aron ◽

Vivekanandan Subramanian ◽

Janet Z. Liu ◽

...

Keyword(s):

Mass Spectrometry ◽

Local Environment ◽

Probiotic Bacterium ◽

Zinc Transporters ◽

Bacterial Metabolism ◽

Dependent Manner ◽

Metal Affinity ◽

Ph Dependent

ABSTRACTZinc is an essential cofactor for bacterial metabolism, and many Enterobacteriaceae express the zinc transporters ZnuABC and ZupT to acquire this metal in the host. Unexpectedly, the probiotic bacterium Escherichia coli Nissle 1917 exhibited appreciable growth in zinc-limited media even when these transporters were deleted. By utilizing in vitro and in vivo studies, as well as native spray metal infusion mass spectrometry and ion identity molecular networking, we discovered that Nissle utilizes yersiniabactin as a zincophore. Indeed, yersiniabactin enables Nissle to scavenge zinc in zinc-limited media, to resist calprotectin-mediated zinc sequestration, and to thrive in the inflamed gut. Moreover, we discovered that yersiniabactin’s affinity for iron or zinc changes in a pH-dependent manner, with higher affinity for zinc as the pH increased. Altogether, we demonstrate that siderophore metal affinity can be influenced by the local environment and reveal a mechanism of zinc acquisition available to many commensal and pathogenic Enterobacteriaceae.

Download Full-text

Lysophosphatidic acid suppresses endothelial cell CD36 expression and promotes angiogenesis via a PKD-1–dependent signaling pathway

Blood ◽

10.1182/blood-2010-12-326017 ◽

2011 ◽

Vol 117 (22) ◽

pp. 6036-6045 ◽

Cited By ~ 28

Author(s):

Bin Ren ◽

James Hale ◽

Sowmya Srikanthan ◽

Roy L. Silverstein

Keyword(s):

Endothelial Cell ◽

Signaling Pathway ◽

Lysophosphatidic Acid ◽

Malignant Tumors ◽

Transcriptional Level ◽

Dependent Manner ◽

Down Regulation ◽

Thrombospondin 1

Abstract In pathologic settings including retinal ischemia and malignant tumors, robust angiogenesis occurs despite the presence in the microenvironment of antiangiogenic proteins containing thrombospondin structural homology (TSR) domains. We hypothesized that antiangiogenesis mediated by TSR-containing proteins could be blunted by localized down-regulation of their cognate receptor on microvascular endothelial cells (MVECs), CD36. Through screening a panel of endothelial cell agonists, we found that lysophosphatidic acid (LPA) dramatically down-regulated CD36 surface expression on primary MVECs. LPA is a lipid-signaling mediator known to have proangiogenic activity, but the mechanisms are largely unknown. We observed that LPA caused CD36 down-regulation in a dose- and time-dependent manner and was long lasting. Down-regulation occurred at the transcriptional level via a signaling pathway involving specific LPA receptors and protein kinase D. LPA-induced MVEC CD36 repression significantly attenuated in vitro antiangiogenic responses to thrombospondin-1, including blockade of migration, tube formation, and VEGFR-2 signaling in response to fibroblast growth factor-2. In vivo relevance was demonstrated by showing that LPA abrogated thrombospondin-1–mediated inhibition of neovascularization of Matrigel plugs implanted in mice. Our data thus indicate that the proangiogenic mechanism of LPA may in part be via switching off the antiangiogenic switch mediated by TSR proteins and CD36.

Download Full-text

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

10.26434/chemrxiv.12213125.v2 ◽

2020 ◽

Cited By ~ 3

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

Entry Efficiency

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Download Full-text

Modulation of Synthetic Tracheal Grafts with Extracellular Matrix Coatings

Bioengineering ◽

10.3390/bioengineering8080116 ◽

2021 ◽

Vol 8 (8) ◽

pp. 116

Author(s):

Lumei Liu ◽

Sayali Dharmadhikari ◽

Robert A. Pouliot ◽

Michael M. Li ◽

Peter M. Minneci ◽

...

Keyword(s):

Mass Spectrometry ◽

Extracellular Matrix ◽

Cell Viability ◽

Dependent Manner ◽

Orthotopic Implantation ◽

Synthetic Scaffolds ◽

Cell Biocompatibility ◽

Dose Dependent

Synthetic scaffolds for the repair of long-segment tracheal defects are hindered by insufficient biocompatibility and poor graft epithelialization. In this study, we determined if extracellular matrix (ECM) coatings improved the biocompatibility and epithelialization of synthetic tracheal grafts (syn-TG). Porcine and human ECM substrates (pECM and hECM) were created through the decellularization and lyophilization of lung tissue. Four concentrations of pECM and hECM coatings on syn-TG were characterized for their effects on scaffold morphologies and on in vitro cell viability and growth. Uncoated and ECM-coated syn-TG were subsequently evaluated in vivo through the orthotopic implantation of segmental grafts or patches. These studies demonstrated that ECM coatings were not cytotoxic and, enhanced the in vitro cell viability and growth on syn-TG in a dose-dependent manner. Mass spectrometry demonstrated that fibrillin, collagen, laminin, and nephronectin were the predominant ECM components transferred onto scaffolds. The in vivo results exhibited similar robust epithelialization of uncoated and coated syn-TG patches; however, the epithelialization remained poor with either uncoated or coated scaffolds in the segmental replacement models. Overall, these findings demonstrated that ECM coatings improve the seeded cell biocompatibility of synthetic scaffolds in vitro; however, they do not improve graft epithelialization in vivo.

Download Full-text

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

10.26434/chemrxiv.12213125 ◽

2020 ◽

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

Entry Efficiency

Download Full-text

Targeting TMPRSS2 and Cathepsin B/L Together May Be Synergistic Against SARS-CoV-2 Infection

10.26434/chemrxiv.12213125.v1 ◽

2020 ◽

Cited By ~ 1

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

Entry Efficiency

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ existing drugs and offers a potential treatment option for COVID-19, currently in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. We showed, analysing our model, that the synergy was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, we found that available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Exploiting the synergy may improve the deployability of drug combinations targeting host proteases required for SARS-CoV-2 entry.

Download Full-text

Estrogen receptor coregulator binding modulator (ERX-11) enhances the activity of CDK4/6 inhibitors against estrogen receptor-positive breast cancers

Breast Cancer Research ◽

10.1186/s13058-019-1227-8 ◽

2019 ◽

Vol 21 (1) ◽

Cited By ~ 1

Author(s):

Suryavathi Viswanadhapalli ◽

Shihong Ma ◽

Gangadhara Reddy Sareddy ◽

Tae-Kyung Lee ◽

Mengxing Li ◽

...

Keyword(s):

Mass Spectrometry ◽

Estrogen Receptor ◽

Combination Therapy ◽

Endocrine Therapy ◽

Tumor Regression ◽

Ex Vivo ◽

Dependent Manner ◽

Dose Dependent

Abstract Background CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. Methods We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. Results ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. Conclusions Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

Download Full-text

Cell surface GRP78 regulates BACE2 via lysosome-dependent manner to maintain mesenchymal phenotype of glioma stem cells

Journal of Experimental & Clinical Cancer Research ◽

10.1186/s13046-020-01807-4 ◽

2021 ◽

Vol 40 (1) ◽

Author(s):

Zihang Chen ◽

Huizhi Wang ◽

Zongpu Zhang ◽

Jianye Xu ◽

Yanhua Qi ◽

...

Keyword(s):

Mass Spectrometry ◽

Stem Cells ◽

Cell Surface ◽

Bioinformatic Analysis ◽

The Cancer Genome Atlas ◽

Glioma Stem Cells ◽

Dependent Manner ◽

Cell Surface Grp78

Abstract Background Glioma stem cells (GSCs) are considered the initial cells of gliomas, contributing to therapeutic resistance. Patient-derived GSCs well recapitulate the heterogeneity of their parent glioma tissues, which can be classified into different subtypes. Likewise, previous works identified GSCs as two distinct subtypes, mesenchymal (MES) and proneural (PN) subtypes, and with general recognition, the MES subtype is considered a more malignant phenotype characterized by high invasion and radioresistance. Therefore, understanding the mechanisms involved in the MES phenotype is necessary for glioblastoma treatment. Methods Data for bioinformatic analysis were obtained from The Cancer Genome Atlas (TCGA) and The Gene Expression Omnibus (GEO) database. An antibody was used to block cell surface glucose-regulated protein 78 (csGRP78). Apoptosis and cell cycle analyses were performed to evaluate radiation damage. Immunofluorescence staining was applied to assess protein expression and distribution. Mass spectrometry combined with bioinformatic analysis was used to screen downstream molecules. Intracranial GSC-derived xenografts were established for in vivo experiments. Results Total GRP78 expression was associated with MES GSC stemness, and csGRP78 was highly expressed in MES GSCs. Targeting csGRP78 suppressed the self-renewal and radioresistance of MES GSCs in vitro and in vivo, accompanied by downregulation of the STAT3, NF-κB and C/EBPβ pathways. Mass spectrometry revealed the potential downstream β-site APP-cleaving enzyme 2 (BACE2), which was regulated by csGRP78 via lysosomal degradation. Knockdown of BACE2 inactivated NF-κB and C/EBPβ and significantly suppressed the tumorigenesis and radioresistance of MES GSCs in vitro and in vivo. Conclusions Cell surface GRP78 was preferentially expressed in MES GSCs and played a pivotal role in MES phenotype maintenance. Thus, blocking csGRP78 in MES GSCs with a high-specificity antibody might be a promising novel therapeutic strategy.

Download Full-text

Targeting TMPRSS2 and Cathepsin B/L together may be synergistic against SARS-CoV-2 infection

PLoS Computational Biology ◽

10.1371/journal.pcbi.1008461 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1008461

Author(s):

Pranesh Padmanabhan ◽

Rajat Desikan ◽

Narendra M. Dixit

Keyword(s):

Cathepsin B ◽

Protein S ◽

Cysteine Proteases ◽

Target Cells ◽

Dependent Manner ◽

Dose Dependent ◽

Protease Expression ◽

Entry Efficiency

The entry of SARS-CoV-2 into target cells requires the activation of its surface spike protein, S, by host proteases. The host serine protease TMPRSS2 and cysteine proteases Cathepsin B/L can activate S, making two independent entry pathways accessible to SARS-CoV-2. Blocking the proteases prevents SARS-CoV-2 entry in vitro. This blockade may be achieved in vivo through ‘repurposing’ drugs, a potential treatment option for COVID-19 that is now in clinical trials. Here, we found, surprisingly, that drugs targeting the two pathways, although independent, could display strong synergy in blocking virus entry. We predicted this synergy first using a mathematical model of SARS-CoV-2 entry and dynamics in vitro. The model considered the two pathways explicitly, let the entry efficiency through a pathway depend on the corresponding protease expression level, which varied across cells, and let inhibitors compromise the efficiency in a dose-dependent manner. The synergy predicted was novel and arose from effects of the drugs at both the single cell and the cell population levels. Validating our predictions, available in vitro data on SARS-CoV-2 and SARS-CoV entry displayed this synergy. Further, analysing the data using our model, we estimated the relative usage of the two pathways and found it to vary widely across cell lines, suggesting that targeting both pathways in vivo may be important and synergistic given the broad tissue tropism of SARS-CoV-2. Our findings provide insights into SARS-CoV-2 entry into target cells and may help improve the deployability of drug combinations targeting host proteases required for the entry.

Download Full-text

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

Protein and Peptide Letters ◽

10.2174/0929866526666190327130037 ◽

2019 ◽

Vol 26 (7) ◽

pp. 494-501 ◽

Cited By ~ 5

Author(s):

Sameer Suresh Bhagyawant ◽

Dakshita Tanaji Narvekar ◽

Neha Gupta ◽

Amita Bhadkaria ◽

Ajay Kumar Gautam ◽

...

Keyword(s):

Biological Effects ◽

Exchange Chromatography ◽

Health Concern ◽

Carbohydrate Binding ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Ic50 Values ◽

Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Download Full-text