Maspin Research Articles

Crystallization and crystallographic studies of human serine protease inhibitor (serpin) B9.

Serine protease inhibitor B9 (serpin B9, also known as protease inhibitor 9 or PI9) plays a critical role in regulating the immune response by specifically inhibiting granzyme B, a serine protease found in cytotoxic T lymphocytes and natural killer cells. Despite its potential as an anticancer drug target, the structural details of serpin B9 have remained elusive until now. In this study, a cleaved form of recombinant human serpin B9 was successfully prepared and crystallized. The crystals belonged to space group P212121, with unit-cell parameters a = 68.51, b = 82.32, c = 101.17 Å, and an X-ray diffraction data set was collected at 1.9 Å resolution. The structure shows that serpin B9 adopts a relaxed conformation, with its cleaved reactive-centre loop inserted into the central β-sheet. Unlike other serpins, serpin B9 shows significant structural deviations around helix D, with a larger surface cavity, which could serve as a promising target for small-molecule inhibitors.

Application of urine proteomics in the diagnosis and treatment effectiveness monitoring of early-stage Mycosis Fungoides

BackgroundMycosis fungoides (MF) is the most common type of cutaneous T cell lymphoma. As the early clinical manifestations of MF are non-specific (e.g., erythema or plaques), it is often misdiagnosed as inflammatory skin conditions (e.g., atopic dermatitis, psoriasis, and pityriasis rosea), resulting in delayed treatment. As there are no effective biological markers for the early detection and management of MF, the aim of the present study was to perform a proteomic analysis of urine samples (as a non-invasive protein source) to identify reliable MF biomarkers.MethodsThirteen patients with early-stage MF were administered a subcutaneous injection of interferon α-2a in combination with phototherapy for 6 months. The urine proteome of patients with early-stage MF before and after treatment was compared against that of healthy controls by liquid chromatography-tandem mass spectrometry. The differentially expressed proteins were subjected to Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Clusters of Orthologous Groups analyses. For validation, the levels of the selected proteins were evaluated by enzyme-linked immunosorbent assay (ELISA).ResultsWe identified 41 differentially expressed proteins (11 overexpressed and 30 underexpressed) between untreated MF patients and healthy control subjects. The proteins were mainly enriched in focal adhesion, endocytosis, and the PI3K-Akt, phospholipase D, MAPK, and calcium signaling pathways. The ELISA results confirmed that the urine levels of Serpin B5, epidermal growth factor (EGF), and Ras homologous gene family member A (RhoA) of untreated MF patients were significantly lower than those of healthy controls. After 6 months of treatment, however, there was no significant difference in the urine levels of Serpin B5, EGF, and RhoA between MF patients and healthy control subjects. The area under the receiver operating characteristic curve values for Serpin B5, EGF, and RhoA were 0.817, 0.900, and 0.933, respectively.ConclusionsThis study showed that urine proteomics represents a valuable tool for the study of MF, as well as identified potential new biomarkers (Serpin B5, EGF, and RhoA), which could be used in its diagnosis and management.

Exogenous serpin B1 restricts immune complex-mediated NET formation via inhibition of a chymotrypsin-like protease and enhances microbial phagocytosis

Immune complex (IC)-driven formation of neutrophil extracellular traps (NETs) is a major contributing factor to the pathogenesis of autoimmune diseases including systemic lupus erythematosus (SLE). Exogenous recombinant human serpin B1 (rhsB1) can regulate NET formation; however, its mechanism(s) of action is currently unknown as is its ability to regulate IC-mediated NET formation and other neutrophil effector functions. To investigate this, we engineered or post-translationally modified rhsB1 proteins that possessed specific neutrophil protease inhibitory activities and pretreated isolated neutrophils with them prior to inducing NET formation with ICs derived from patients with SLE, PMA, or the calcium ionophore A23187. Neutrophil activation and phagocytosis assays were also performed with rhsB1 pretreated and IC-activated neutrophils. rhsB1 dose-dependently inhibited NET formation by all three agents in a process dependent on its chymotrypsin-like inhibitory activity, most likely cathepsin G. Only one variant (rhsB1 C344A) increased surface levels of neutrophil adhesion/activation markers on IC-activated neutrophils and boosted intracellular ROS production. Further, rhsB1 enhanced complement-mediated neutrophil phagocytosis of opsonized bacteria but not ICs. In conclusion, we have identified a novel mechanism of action by which exogenously administered rhsB1 inhibits IC, PMA, and A2138-mediated NET formation. Cathepsin G is a well-known contributor to autoimmune disease but to our knowledge, this is the first report implicating it as a potential driver of NET formation. We identified the rhsB1 C334A variant as a candidate protein that can suppress IC-mediated NET formation, boost microbial phagocytosis, and potentially impact additional neutrophil effector functions including ROS-mediated microbial killing in phagolysosomes.

Overexpression of an Engineered SERPINB9 Enhances Allogeneic T-cell Persistence and Efficacy.

Allogeneic chimeric antigen receptor (CAR)-expressing T cells offer many advantages over autologous therapies, but their benefits are curtailed by graft-versus-host disease and elimination by recipient immune cells. Moreover, just as with autologous therapies, allogeneic CAR T cells are susceptible to activation-induced cell death (AICD) caused by chronic antigen exposure (CAE). Granzyme B- and Fas/Fas ligand-initiated caspase-mediated apoptoses are key mechanisms of T-cell death caused by T/NK cell-mediated allorejection or CAE. We explored a protective strategy of engineering CAR T cells to overexpress variants of the Granzyme B-specific serine protease inhibitor SERPINB9 (SB9) to improve allogeneic T-cell persistence and antitumor efficacy. We showed that the overexpression of an SB9 variant with broadened caspase specificity, SB9(CAS), not only significantly reduced rejection of allogeneic CAR T cells but also increased their resistance to AICD and enabled them to thrive better under CAE, thus improving allogeneic T-cell persistence and antitumor activity in vitro and in vivo. In addition, although SB9(CAS) overexpression improved the efficacy of allogeneic CAR T-cell therapy by conferring protection to cell death, we did not observe any autonomous growth, and the engineered CAR T cells were still susceptible to an inducible suicide switch. Hence, SB9(CAS) overexpression is a promising strategy that can strengthen current development of cell therapies, broadening their applications to address unmet medical needs.

Quantitative Proteomics of COVID-19 Recovered Patients Identifies Long-Term Changes in Sperm Proteins Leading to Cellular Stress in Spermatozoa.

Following an initial recovery, COVID-19 survivors struggle with a spectrum of persistent medical complications, including fatigue, breathlessness, weight loss, hair loss, and attention deficits. Additionally, there is growing evidence of adverse effects of COVID-19 on the male reproductive system. This investigation seeks to understand the long-term ramifications on male fertility by examining hormonal profiles, semen parameters, and sperm proteome of recovered COVID-19 patients compared to controls. The serum hormone profiles between the two groups showed minimal variations except for prolactin, cortisol, and testosterone levels. Testosterone levels were slightly lower, while prolactin and cortisol were elevated in COVID-19 cases compared to controls. Though semen parameters exhibited no significant disparities between the COVID-19 and control groups, quantitative proteomics analysis revealed changes in sperm proteins. It identified 190 differentially expressed proteins, of which 161 were upregulated and 29 downregulated in COVID-19 cases. Western blotting analysis validated the differential expression of serpin B4 and calpain 2. Bioinformatics analysis signifies cellular stress in the spermatozoa of COVID-19 recovered patients and thus, SOD and MDA levels in semen were measured. MDA levels were found to be significantly elevated, indicating lipid peroxidation in COVID-19 samples. While the effects of COVID-19 on semen parameters may exhibit a potential for reversal within a short duration, the alterations it inflicts on sperm proteome are persisting consequences on male fertility. This study paves the path for further research and emphasizes the significance of comprehending the complex molecular processes underlying the long-term consequences of COVID-19 on male reproductive health.

Increased SERPINB2 potentiates 15LO1 expression via STAT6 signalling in epithelial cells in eosinophilic chronic rhinosinusitis with nasal polyps.

SERPINB2, a biomarker of Type-2 (T2) inflammatory processes, has been described in the context of asthma. Chronic rhinosinusitis with nasal polyps (CRSwNP) is also correlated with T2 inflammation and elevated 15LO1 induced by IL-4/13 in nasal epithelial cells. The aim of this study was to evaluate the expression and location of SERPINB2 in nasal epithelial cells (NECs) and determine whether SERPINB2 regulates 15LO1 and downstream T2 markers in NECs via STAT6 signalling. SERPINB2 gene expression in bulk and single-cell RNAseq database was analysed by bioinformatics analysis. SERPINB2, 15LO1 and other T2 markers were evaluated from CRSwNP and HCs NECs. The colocalization of SERPINB2 and 15LO1 was evaluated by immunofluorescence. Fresh NECs were cultured at an air-liquid interface with or without IL-13, SERPINB2 Dicer-substrate short interfering RNAs (DsiRNAs) transfection, exogenous SERPINB2, 15-HETE recombinant protein and pSTAT6 inhibitors. 15LO1, 15-HETE and downstream T2 markers were analysed by qRT-PCR, western blot and ELISA. SERPINB2 expression was increased in eosinophilic nasal polyps compared with that in noneosinophilic nasal polyps and control tissues and positively correlated with 15LO1 and other downstream T2 markers. SERPINB2 was predominantly expressed by epithelial cells in NP tissue and was colocalized with 15LO1. In primary NECs invitro, SERPINB2 expression was induced by IL-13. Knockdown or overexpression SERPINB2 decreased or enhanced expression of 15LO1 and 15-HETE in NECs, respectively, in a STAT6-dependent manner. SERPINB2 siRNA also inhibited the expression of the 15LO1 downstream genes, such as CCL26, POSTN and NOS2. STAT6 inhibition similarly decreased SERPINB2-induced 15LO1. SERPINB2 is increased in NP epithelial cells of eosinophilic CRSwNP (eCRSwNP) and contributes to T2 inflammation via STAT6 signalling. SERPINB2 could be considered a novel therapeutic target for eCRSwNP.

A meta‑ and bioinformatics analysis of maspin expression levels influencing the prognosis of patients with breast cancer.

Maspin is a serine protease inhibitor that is encoded by the human SERPINB5 gene. As a tumor inhibitor, it can inhibit the growth of tumor cells, increase adhesion between tumor cells and inhibit tumor angiogenesis. In the present study, a meta- and bioinformatics analysis was performed through the PubMed and China National Knowledge Infrastructure databases including entries added until up to March 20, 2023. It was found that compared with normal breast tissue, maspin expression was downregulated in breast cancer tissue. Maspin expression was negatively associated with lymph node metastasis. According to Kaplan-Meier plotter, it was found that lower maspin expression was negatively associated with the overall and distant metastasis-free survival rate of patients with estrogen receptor-positive, luminal A and grade 2 breast cancer. High expression of maspin was also positively associated with the relapse-free survival rate of patients of the luminal A subtype. Low maspin expression was positively associated with the post-progression and distant metastasis-free survival rate of the progesterone receptor-negative subtype. According to the GEPIA database, SERPINB5 mRNA expression was higher in normal than breast cancer tissues and negatively correlated with the TNM stage. High expression of maspin was also positively associated with the overall survival rate. In the UALCAN database, it was found that the mRNA and promoter methylation levels of SERPINB5 were higher in normal than in breast cancer tissues. These findings suggest that the expression of maspin may serve as a potential marker to indicate the occurrence, subsequent progression and even prognosis of breast cancer.

Mass Spectrometry-Based Proteomics Identifies Serpin B9 as a Key Protein in Promoting Bone Metastases in Lung Cancer.

SB9 as a therapeutic target for LCBM.

Recombinant streptococcal protein G-modified metal-organic framework ZIF-8 for the highly selective purification of immunoglobulin G from human serum

A novel solid phase extractant His-rSPG@ZIF-8 was prepared by covalently coupling recombinant streptococcal protein G (His-rSPG) with ZIF-8. The His-rSPG@ZIF-8 composite was characterized by Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy (Raman), X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Due to the specific binding between the immunoglobulin binding region of His-rSPG and the Fc region of immunoglobulin G (IgG), the His-rSPG@ZIF-8 composite demonstrated exceptional selectivity in adsorbing IgG. In Britton-Robinson buffer (BR buffer) with a salt concentration of 500 mmol L−1 (0.04 mol L−1, pH 8.0), the His-rSPG@ZIF-8 composite exhibited a remarkable adsorption efficiency of 99.8 % for 0.05 mg of the composite on 200 μL of IgG solution (100 μg mL−1). The adsorption behavior of the His-rSPG@ZIF-8 composite aligns with the Langmuir adsorption model, and the theoretical maximum adsorption capacity is 1428.6 mg g−1. The adsorbed IgG molecules were successfully eluted using a SDS solution (0.5 %, m/m), resulting in a recovery rate of 91.2 %. Indeed, the His-rSPG@ZIF-8 composite was successfully utilized for the isolation and purification of IgG from human serum samples. The obtained IgG exhibited high purity, as confirmed by SDS-PAGE analysis. Additionally, LC-MS/MS analysis was employed to identify the human serum proteins following the adsorption and elution process using the His-rSPG@ZIF-8 composite material. The results revealed that the recovered solution contained an impressive content of immunoglobulin, accounting for 62.4 % of the total protein content. Furthermore, this process also led to the significant enrichment of low abundance proteins such as Serpin B4 and Cofilin-1. Consequently, the His-rSPG@ZIF-8 composite holds great promise for applications such as IgG purification and immunoassays. At the same time, it expands the application of metal-organic frameworks in the field of proteomics.

Differentiation between Gastric and Colorectal Adenocarcinomas Based on Maspin, MLH1, PMS2 and K-Ras Concentrations Determined Using Stochastic Sensors

Background: Gastrointestinal adenocarcinomas are a worldwide and some of the most important causes of death related to cancers. MLH1, PMS2, and K-Ras are some of the main molecules responsible for the control of cellular proliferation. They are widely used as biomarkers for the evaluation of the features of tumoral processes and the clinicopathological characteristics. They depend on the type of cells implied in the tumoral process, and it can be observed in the concentrations of them in different biological fluids. Maspin, also known as peptidase inhibitor 5 or serpin B5 is a tumor suppressor which inhibits invasion and angiogenesis and also regulates apoptosis, but it can also present oncogenic activity depending on tumor location and histology and on the subcellular maspin localization. Its correlations with gastric and colorectal carcinomas have been emphasized in a series of articles, and in this work, a method is used to quantify the concentrations of maspin in three biological fluids, allowing correlations with pathological features. Methods: Patients with their clinical and pathological features were selected from the database of the project GRAPHSENSGASTROINTES and used accordingly with the Ethics committee approval nr. 32647/2018 awarded by the County Emergency Hospital from Targu-Mures. Three kinds of samples have been analyzed (saliva, whole blood, and urine) using a stochastic method using stochastic microsensors. Results: The results obtained using stochastic sensors were correlated with the location of cancer, and there have been elaborated a series of criteria to differentiate gastric cancers from colorectal ones. Conclusions: There can be differentiation between the two types of cancers by using the concentrations of MLH1, PMS2, and K-Ras in saliva and urine samples or the levels of maspin in whole blood and urine or in whole blood, urine, and saliva. The data analysis led to a series of criteria for evaluation of the cancer location. Using only MLH1 and PMS2 concentrations in one of the two kinds of samples was only indicative and did not cover most cases. The use of the criteria only for MLH1 and PMS2 increased the probability of finding out the location, but the best results require the concentrations of K-Ras in the two kinds of samples as additional criteria.

Circular RNA sequencing identified circARNTL2 as a pathogenic factor in psoriasis by facilitating proliferation and cell cycle progression of keratinocytes

Psoriasis is a chronic recurrent skin disease, with excessive proliferation of keratinocytes. Recent studies indicated the pathogenic roles of circular RNA (circRNA) in psoriasis. Here, we screened the circRNA profiles from five psoriatic skin lesions and five normal skin tissues by circRNA sequencing and identified 1118 differentially expressed circRNAs (DECs) between psoriatic and normal groups. Among these DECs, high abundant circARNTL2 has been proven upregulated in psoriatic skin lesions by RT-qPCR assay. Then, the head-to-tail structure of circARNTL2 was validated by Sanger sequencing and RNase R digestion assay. Moreover, we determined cytoplastic location of circARNTL2 by RT-qPCR assay of nuclear/cytoplasmic RNA and FISH analysis. Further experiments demonstrated that silencing circARNTL2 expression could block cell proliferation and cell cycle progression of keratinocytes. Mechanistically, circARNTL2 can bind to and regulate Serpin B4 which also affects the proliferation of keratinocytes. These findings provide evidence for the role of circARNTL2 in psoriasis.

PROTEOMIC PROFILING IDENTIFIES GRANZYME B INHIBITOR SERPIN B9 AS MEDIATOR OF RESISTANCE TO CAR T‐CELL AND BISPECIFIC ANTIBODY TREATMENT IN NODAL B‐CELL LYMPHOMA

Introduction: Treatment options for relapsed or refractory (r/r) B-cell non-Hodgkin lymphomas (B-NHL) have broadened towards T-cell engaging therapies, including CD19-targeting chimeric antigen receptor T-cells (CD19-CAR) and bispecific antibodies (CD19-BsAb). Although CD19-CAR and CD19-BsAb induce durable responses in some r/r B-NHL patients, response remains heterogenous and a significant proportion of patients experience insufficient responses or relapse. A thorough understanding of tumor-intrinsic mechanisms of resistance is crucial for the clinical improvement of T-cell immunotherapy. Methods: Aiming to identify lymphoma cell-inherent mechanisms that impair response to T-cell engaging therapy, we quantified the in-vitro response of 46 B-NHL cell lines to 3rd generation CD19-CAR and CD19-BsAb. Response was measured using a high-throughput flow cytometry-based assay. In parallel, cell lines were subjected to proteomic profiling and protein abundance was regressed on in-vitro response in order to identify proteomic determinants of response. We further combined CD19-CAR treatment with clinically relevant drugs and assessed how killing of lymphoma cells and expansion of T-cells was altered. Results: Response to CD19-CAR and CD19-BsAb was highly heterogeneous across cell lines and B-NHL entities, but not significantly linked to their proliferation rate or CD19 expression. Integration of the proteomic profiles revealed that abundance of Serpin B9, endogenous inhibitor of T-cell effector molecule granzyme B, was associated with poor response to CD19-CAR and CD19-BsAb. This finding was validated in overexpression and knock-out models. Overexpression of SERPINB9 indeed attenuated response to CD19-CAR and CD19-BsAb, while its knock-out rendered cell lines more sensitive. The observed changes in susceptibility to T-cell-mediated cytotoxicity upon modulation of SERPINB9 expression were independent of T-cell donor and expression of T-cell activation and proliferation markers. We sought to improve T-cell-mediated cytotoxicity specifically in samples with high SERPINB9 expression and found that immune checkpoint inhibitors and lenalidomide enhanced expansion of CAR T-cells and increased killing of lymphoma cells. In addition, vorinostat and antibody-drug-conjugate polatuzumab vedotin specifically killed lymphoma cells without affecting T-cell expansion. However, the observed improvement in T-cell-mediated cytotoxicity was independent of SERPINB9 expression. Keywords: aggressive B-cell non-Hodgkin lymphoma, immunotherapy, indolent non-Hodgkin lymphoma No conflicts of interests pertinent to the abstract.

Abstract 3936: ALK fusion oncogene driven SERPINB4 expression enhances tumor survival in NSCLC

Abstract Anaplastic lymphoma kinase (ALK) fusion variants in non-small-cell-lung cancer (NSCLC) consist of numerous dimerising fusion partners, with the most common being EML4. Clinical data suggests that the degree of treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs among the variant present in the patient tumor. Therefore, a better understanding the oncogenic signaling networks driven by different ALK-fusion variants is important. Here, we developed highly controlled doxycycline-inducible cell models bearing four different ALK fusion proteins, namely EML4-ALK-V1, EML4-ALK-V3, KIF5B-ALK, and TFG-ALK, in the context of non-tumorigenic NL20 human bronchial epithelial cells. These were complimented by patient-derived NSCLC cell lines harboring either EML4-ALK-V1 or EML4-ALK-V3 fusions. RNA-seq and phosphoproteomics analysis were employed to identify dysregulated genes and hyper/hypo-phosphorylated proteins associated with ALK fusion expression. Among ALK fusion induced responses, we noted a robust inflammatory signature that included up-regulation of the Serpin B4 serine protease inhibitor in both NL20-inducible cell models and ALK-positive NSCLC patient-derived cell lines. We show that STAT3 is a major transcriptional regulator of SERPINB4 downstream of ALK fusions, along with NF-kB and AP1. The upregulation of SERPINB4 promotes survival of ALK fusion expressing cells and inhibits natural killer (NK) cell-mediated cytotoxicity. In conclusion, our study reveals a novel ALK downstream survival axis that regulates Serpin B4 expression and identifies a molecular target that has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC. Citation Format: Tzu-Po Chuang, Wei-Yun Lai, Jonatan L. Gabre, Dan E. Lind, Ganesh Umapathy, Abdulmalik A. Bokhari, Bengt Bergman, Linnea Kristenson, Fredrik B. Thorén, Anh Le, Robert Doebele, Jimmy V. Eynden, Ruth H. Palmer, Bengt Hallberg. ALK fusion oncogene driven SERPINB4 expression enhances tumor survival in NSCLC. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3936.

Potential biomarker proteins for aspiration pneumonia detected by shotgun proteomics using buccal mucosa samples: a cross-sectional case–control study

BackgroundAspiration pneumonia (AP), which is a major cause of death in the elderly, does present with typical symptoms in the early stages of onset, thus it is difficult to detect and treat at an early stage. In this study, we identified biomarkers that are useful for the detection of AP and focused on salivary proteins, which may be collected non-invasively. Because expectorating saliva is often difficult for elderly people, we collected salivary proteins from the buccal mucosa.MethodsWe collected samples from the buccal mucosa of six patients with AP and six control patients (no AP) in an acute-care hospital. Following protein precipitation using trichloroacetic acid and washing with acetone, the samples were analyzed by liquid chromatography and tandem mass spectrometry (LC–MS/MS). We also determined the levels of cytokines and chemokines in non-precipitated samples from buccal mucosa.ResultsComparative quantitative analysis of LC–MS/MS spectra revealed 55 highly (P values < 0.10) abundant proteins with high FDR confidence (q values < 0.01) and high coverage (> 50%) in the AP group compared with the control group. Among the 55 proteins, the protein abundances of four proteins (protein S100-A7A, eukaryotic translation initiation factor 1, Serpin B4, and peptidoglycan recognition protein 1) in the AP group showed a negative correlation with the time post-onset; these proteins are promising AP biomarker candidates. In addition, the abundance of C-reactive protein (CRP) in oral samples was highly correlated with serum CRP levels, suggesting that oral CRP levels may be used as a surrogate to predict serum CRP in AP patients. A multiplex cytokine/chemokine assay revealed that MCP-1 tended to be low, indicating unresponsiveness of MCP-1 and its downstream immune pathways in AP.ConclusionOur findings suggest that oral salivary proteins, which are obtained non-invasively, can be utilized for the detection of AP.

ALK fusion NSCLC oncogenes promote survival and inhibit NK cell responses via SERPINB4 expression

Anaplastic lymphoma kinase (ALK) fusion variants in Non-Small Cell Lung Cancer (NSCLC) consist of numerous dimerizing fusion partners. Retrospective investigations suggest that treatment benefit in response to ALK tyrosine kinase inhibitors (TKIs) differs dependent on the fusion variant present in the patient tumor. Therefore, understanding the oncogenic signaling networks driven by different ALK fusion variants is important. To do this, we developed controlled inducible cell models expressing either Echinoderm Microtubule Associated Protein Like 4 (EML4)-ALK-V1, EML4-ALK-V3, Kinesin Family Member 5B (KIF5B)-ALK, or TRK-fused gene (TFG)-ALK and investigated their transcriptomic and proteomic responses to ALK activity modulation together with patient-derived ALK-positive NSCLC cell lines. This allowed identification of both common and isoform-specific responses downstream of these four ALK fusions. An inflammatory signature that included upregulation of the Serpin B4 serine protease inhibitor was observed in both ALK fusion inducible and patient-derived cells. We show that Signal transducer and activator of transcription 3 (STAT3), Nuclear Factor Kappa B (NF-κB) and Activator protein 1 (AP1) are major transcriptional regulators of SERPINB4 downstream of ALK fusions. Upregulation of SERPINB4 promotes survival and inhibits natural killer cell-mediated cytotoxicity, which has potential for therapeutic impact targeting the immune response together with ALK TKIs in NSCLC.

Quantitative Proteomic Analysis of Non-Tobacco Associated Oral Squamous Cell Carcinoma Reveals Deregulation of Cytoskeletal and Apoptotic Proteins

The exact etiology of non-tobacco associated oral squamous cell carcinoma (NT-OSCC) is still unknown. The lack of established biomarkers for oral NT-OSCC has resulted in less effective management and poor prognosis. Here, we report for the first time a panel of potential markers identified from the quantitative proteomic analysis of NT-OSCC using two-dimensional gel-electrophoresis (2D-GE) using matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF) coupled with mass spectrometry (MS) and further analysis using protein analysis through evolutionary relationships (PANTHER) database. To quantitatively analyze the proteomic profile of non-tobacco associated oral squamous cell carcinoma. Twenty fresh tissue samples were collected from healthy controls and NT-OSCC, ten each, and were subjected to proteomic analysis. Sample quantification for the presence of protein was done using Bradford assay and bovine serum albumin was used as a standard protein to obtain the standard graph. Fractionation of protein was done using sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE) and they were separated based on their molecular weight. MS analysis was done and the purified peptides were analysed using MALDI-TOF. PANTHER database for functional classification and pathway analysis was done for identification of protein expression. Our approach of combining 2D-GE with MS identified four candidate proteins including keratin, alpha-1-antitrypsin (AAT), S100 and serpin B5 with significant differential expression in NT-OSCC as compared with healthy controls. The results showed that the levels of these proteins were significantly upregulated in NT-OSCC when compared to the healthy controls that suggests that these proteins can be used as candidate targets for NT-OSCC therapeutics. The differentially expressed proteins are found to be involved in apoptotic signalling pathways, cytoskeletal dynamics and are known to play a critical role in oral tumorigenesis. Put together, the results provide available baseline information for understanding the development and progression of NT-OSCC. These identified proteins on further validation may be used as potential biomarkers in future for early detection and predicting therapeutic outcome of patients with NT-OSCC.

Pre-clinical evaluation of antiproteases as potential candidates for HIV-1 pre-exposure prophylaxis.

Previous studies on highly HIV-1-exposed, yet persistently seronegative women from the Punwami Sex Worker cohort in Kenya, have shed light on putative protective mechanisms, suggesting that mucosal immunological factors, such as antiproteases, could be mediating resistance to HIV-1 transmission in the female reproductive tract. Nine protease inhibitors were selected for this study: serpin B4, serpin A1, serpin A3, serpin C1, cystatin A, cystatin B, serpin B13, serpin B1 and α-2-macroglobulin-like-protein 1. We assessed in a pilot study, the activity of these antiproteases with cellular assays and an ex vivo HIV-1 challenge model of human ecto-cervical tissue explants. Preliminary findings with both models, cellular and tissue explants, established an order of inhibitory potency for the mucosal proteins as candidates for pre-exposure prophylaxis when mimicking pre-coital use. Combination of all antiproteases considered in this study was more active than any of the individual mucosal proteins. Furthermore, the migration of cells out of ecto-cervical explants was blocked indicating potential prevention of viral dissemination following amplification of the founder population. These findings constitute the base for further development of these mucosal protease inhibitors for prevention strategies.

Distinct proteomic profile of ovarian follicular fluid in ewes from small versus large developing follicles

This study aimed to describe the proteins of the ovarian follicular fluid from ewes. The follicular fluid was taken from 294 ovaries collected from slaughterhouses and split into two groups based on follicle size: Group 1 (< 4 mm); and Group 2 (≥ 4 to 6 mm). The samples for ESI-Q-TOF MS/MS mass spectrometry were prepared through Tryptic in-gel digestion. For comparisons between groups, the data were analyzed using uni and multivariate statistical analysis, represented by principal component analysis (PCA), the measure of a variable's importance (VIP score) in the partial least squares-discriminant analysis (PLS-DA), the fold change, and t-test, using the exponentially modified protein abundance index (emPAI). Moreover, gene ontology was performed to identify molecular function, biological process, and cellular component. Fifty-five major proteins were identified in the 2 groups. The separation of the formed clusters was clearly observed in the PCA. Twelve proteins had a VIP score with α ≥ 1.5. Only 2 proteins were found in higher abundance in Group 1, the serum amyloid A-4 protein isoform X1 and trinucleotide repeat-containing gene 6c protein isoform X2. However, in Group 2, 10 proteins were in higher abundance: complement component C6, serotransferrin isoform X2, fibrinopeptide A, tetranectin, inter-alpha-trypsin inhibitor heavy chain H4 isoform X1, serpin B9 isoform X2, alpha-enolase isoform X6, serum paraoxonase/arylesterase 1, complement factor B, and complement C3 isoform X2, partial. We concluded, therefore, that a distinct profile of protein abundance is observed in small vs large growing follicles, including proteins especially related to ovulation, follicular metabolism, and oocyte development and quality.

Semisolid Wet Sol–Gel Silica/Hydroxypropyl Methyl Cellulose Formulation for Slow Release of Serpin B3 Promotes Wound Healing In Vivo

Foot ulcerations are a disabling complication of diabetes and no treatment is currently available based on disease mechanisms. The protein serpin B3 (SB3) was identified as a positive biomarker of successful diabetic wound healing; therefore, its exogenous administration may promote healing. The topical administration of SB3 is challenging due to its protein nature. Physical entrapment in wet sol–gel silica can stabilize the protein’s conformation and permit its sustained delivery. However, irreversible syneresis and poor viscoelastic properties hamper wet sol–gel silica application as a semisolid vehicle. To overcome these limits, a sol–gel silica/hydroxypropylmethylcellulose (HPMC) hydrogel blend was developed. SB3 entrapped in 8% SiO2 wet sol–gel silica preserved its structure, was stabilized against denaturation, and was slowly released for at least three days. Blending a silica gel with an HPMC–glycerol (metolose-G) hydrogel permitted spreadability without affecting the protein’s release kinetics. When administered in vivo, SB3 in silica/metolose-G—but not in solution or in metolose-G alone—accelerated wound healing in SB3 knockout and diabetic mouse models. The results confirmed that SB3 is a new pharmacological option for the treatment of chronic ulcers, especially when formulated in a slow-releasing vehicle. Silica–metolose-G represents a novel type of semisolid dosage form which could also be applied for the formulation of other bioactive proteins.

Transcriptomic Analysis of Human Keratinocytes Treated with Galactomyces Ferment Filtrate, a Beneficial Cosmetic Ingredient.

Galactomyces ferment filtrate (GFF, Pitera™) is a cosmetic ingredient known to have multiple skin care benefits, such as reducing redness and pore size via the topical application of its moisturizer form. Although GFF is known to act partly as an antioxidative agonist for the aryl hydrocarbon receptor (AHR), its significance in keratinocyte biology is not fully understood. In this study, we conducted a transcriptomic analysis of GFF-treated human keratinocytes. Three different lots of GFF consistently modulated 99 (22 upregulated and 77 downregulated) genes, including upregulating cytochrome P450 1A1 (CYP1A1), a specific downstream gene for AHR activation. GFF also enhanced the expression of epidermal differentiation/barrier-related genes, such as small proline-rich proteins 1A and 1B (SPRR1A and SPRR1B), as well as wound healing-related genes such as serpin B2 (SERPINB2). Genes encoding components of tight junctions claudin-1 (CLDN1) and claudin-4 (CLDN4) were also target genes upregulated in the GFF-treated keratinocytes. In contrast, the three lots of GFF consistently downregulated the expression of inflammation-related genes such as chemokine (C-X-C motif) ligand 14 (CXCL14) and interleukin-6 receptor (IL6R). These results highlight the beneficial properties of GFF in maintaining keratinocyte homeostasis.