Maspin Research Articles

PAI-2/SerpinB2 inhibits proteolytic activity in a P. gingivalis-dominated multispecies bacterial consortium

ObjectiveThe aim of this study was to investigate the ability of the serine protease inhibitor plasminogen activator inhibitor type 2 (PAI-2/Serpin B2) to inhibit proteases produced by a multispecies bacterial consortium in vitro. BackgroundGingival and periodontal inflammation is associated with an increased flow of protein-rich gingival fluid. This nutritional change in the microenvironment favors bacteria with a proteolytic phenotype, triggering inflammation and associated tissue breakdown. PAI-2 is produced by macrophages and keratinocytes and is present in very high concentrations in gingival crevicular fluid; the highest level in the body. DesignA multispecies bacterial consortium comprising nine bacterial strains, resembling the conditions in a periodontal pocket, was grown planktonically and as a biofilm. After seven days PAI-2 was added to the consortium and the proteolytic activity was assayed with fluorogenic protease substrates; FITC-labeled casein to detect global protease activity, fluorescent H-Gly-Pro-AMC for serine protease activity and fluorescent BIKKAM-10 for Porphyromonas gingivalis-associated protease activity. Protease activity associated with biofilm cells was examined by confocal scanning laser microscopy. ResultsPAI-2 inhibited proteolytic activity of the bacterial consortium, as seen by decreased fluorescence of all substrates. PAI-2 specifically inhibited P. gingivalis proteolytic activity. ConclusionTo our knowledge, this is the first time that PAI-2 has been shown to inhibit bacterial proteases. Given the high concentration of PAI-2 in the gingival region, our results indicate that PAI-2 might play a role for the integrity of the epithelial barrier.

Imaging of Angiotropism/Vascular Co-Option in a Murine Model of Brain Melanoma: Implications for Melanoma Progression along Extravascular Pathways.

Angiotropism/pericytic mimicry and vascular co-option involve tumor cell interactions with the abluminal vascular surface. These two phenomena may be closely related. However, investigations of the two processes have developed in an independent fashion and different explanations offered as to their biological nature. Angiotropism describes the propensity of tumor cells to spread distantly via continuous migration along abluminal vascular surfaces, or extravascular migratory metastasis (EVMM). Vascular co-option has been proposed as an alternative mechanism by which tumors cells may gain access to a blood supply. We have used a murine brain melanoma model to analyze the interactions of GFP human melanoma cells injected into the mouse brain with red fluorescent lectin-labeled microvascular channels. Results have shown a striking spread of melanoma cells along preexisting microvascular channels and features of both vascular co-option and angiotropism/pericytic mimicry. This study has also documented the perivascular expression of Serpin B2 by angiotropic melanoma cells in the murine brain and in human melanoma brain metastases. Our findings suggest that vascular co-option and angiotropism/pericytic mimicry are closely related if not identical processes. Further studies are needed in order to establish whether EVMM is an alternative form of cancer metastasis in addition to intravascular cancer dissemination.

Neutrophil elastase‐deficient mice form neutrophil extracellular traps in an experimental model of deep vein thrombosis

ESSENTIALS: Neutrophil elastase (NE) plays a role in extracellular trap formation (NETosis) triggered by microbes. The contribution of NE was evaluated in mouse NETosis models of sterile inflammation and thrombosis. NE is not required for mouse neutrophil NET production in vitro with non-infectious stimuli. NE deficiency had no significant effect on thrombosis in the inferior vena cava stenosis model. Neutrophil serine proteases have been implicated in coagulation and neutrophil extracellular trap (NET) formation. In human neutrophils, neutrophil elastase (NE) translocates to the nucleus during NETosis and cleaves histones, thus aiding in chromatin decondensation. NE(-/-) mice were shown not to release NETs in response to microbes. However, mouse studies evaluating the role of NE in NET formation in sterile inflammation and thrombosis are lacking. We wished to establish if neutrophils from NE(-/-) mice have a defect in NETosis, similar to peptidylarginine deiminase 4 (PAD4(-/-)) mice, and how this might have an impact on venous thrombosis, a model where NETs are produced and are crucial to thrombus development. We performed in vitro NET assays using neutrophils from wild-type (WT), NE(-/-), SerpinB1 (SB1)(-/-) and NE(-/-) SB1(-/-) mice. We compared WT and NE(-/-) animals using the inferior vena cava stenosis model of deep vein thrombosis (DVT). Neutrophil elastase deficiency resulted in a small reduction in ionomycin-induced NET formation in vitro without affecting histone citrullination. However, NET production in response to phorbol 12-myristate 13-acetate or platelet activating factor was normal in neutrophils from two independent NE-deficient mouse lines, and in NE(-/-) SB1(-/-) as compared with SB1(-/-) neutrophils. NE deficiency or inhibition did not prevent NETosis in vivo or DVT outcome. Neutrophil elastase is not required for NET formation in mice. NE(-/-) mice, which form pathological venous thrombi containing NETs, do not phenocopy PAD4(-/-) mice in in vitro NETosis assays or experimental venous thrombosis. Our study suggests that NET-targeted therapies need to be highly effective to have an impact on DVT.

A novel fully-human cytolytic fusion protein based on granzyme B shows in vitro cytotoxicity and ex vivo binding to solid tumors overexpressing the epidermal growth factor receptor

Human cytolytic fusion proteins (hCFPs) offer a promising immunotherapeutic approach for the treatment of solid tumors, avoiding the immunogenicity and undesirable side-effects caused by immunotoxins derived from plants or bacteria. The well-characterized human serine protease granzyme B has already been used as a therapeutic pro-apoptotic effector domain. We therefore developed a novel recombinant hCFP (GbR201K-scFv1711) consisting of an epidermal growth factor receptor-specific human antibody fragment and a granzyme B point mutant (R201K) that is insensitive to serpin B9 (PI9), a natural inhibitor of wild-type granzyme B that is often expressed in solid tumors. We found that GbR201K-scFv1711 selectively bound to epidermoid cancer and rhabdomyosarcoma cells and was rapidly internalized by them. Nanomolar concentrations of GbR201K-scFv1711 achieved the specific killing of epidermoid cancer cells by inducing apoptosis, and similar effects were observed in rhabdomyosarcoma cells when GbR201K-scFv1711 was combined with the endosomolytic substance chloroquine. The novel hCFP was stable in serum and bound to human rhabdomyosarcoma tissue ex vivo. These data confirm that GbR201K-scFv1711 is a promising therapeutic candidate suitable for further clinical investigation.

Epidermal thymic stromal lymphopoietin predicts the development of atopic dermatitis during infancy

Epidermal thymic stromal lymphopoietin predicts the development of atopic dermatitis during infancy

Granzyme B-based cytolytic fusion protein targeting EpCAM specifically kills triple negative breast cancer cells in vitro and inhibits tumor growth in a subcutaneous mouse tumor model

Triple-negative breast cancer (TNBC) is associated with poor prognosis and high prevalence among young premenopausal women. Unlike in other breast cancer subtypes, no targeted therapy is currently available. Overexpression of epithelial cell adhesion molecule (EpCAM) in 60% of TNBC tumors correlates with poorer prognosis and is associated with cancer stem cell phenotype. Thus, selective elimination of EpCAM+ TNBC tumor cells is of clinical importance.Therefore, we constructed a fully human targeted cytolytic fusion protein, designated GbR201K-αEpCAM(scFv), in which an EpCAM-selective single-chain antibody fragment (scFv) is genetically fused to a granzyme B (Gb) mutant with reduced sensitivity to its natural inhibitor serpin B9. In vitro studies confirmed its specific binding, internalization and cytotoxicity toward a panel of EpCAM-expressing TNBC cells. Biodistribution kinetics and tumor-targeting efficacy using MDA-MB-468 cells in a human TNBC xenograft model in mice revealed selective accumulation of GbR201K-αEpCAM(scFv) in the tumors after i.v. injection. Moreover, treatment of tumor-bearing mice demonstrated a prominent inhibition of tumor growth of up to 50 % in this proof-of-concept study. Taken together, our results indicate that GbR201K-αEpCAM(scFv) is a promising novel targeted therapeutic for the treatment of TNBC.

Proteome Analysis the Differences of the Mesenchymal Stem Cells and Extracorporeal Shock Wave Therapy Enhancing Diabetic Wound Healing

PURPOSE: Non-healing foot ulcers in patients with diabetes are the leading cause of complications. Our studies have demonstrated topical application of bone marrow-derived mesenchymal stem cells (MSCs) and extracorporeal shock wave therapy (ESWT) both could enhance the diabetic wound healing in a rodent STZ-induced diabetes model.1,2 However, the details mechanisms how MSCs and ESWT increased the wound healing is still unclear. Therefore, this study investigated the topical wounding margin tissue between the MSC group and ESWT group and diabetic controls by using 2D proteome study. MATERIALS AND METHODS: A dorsal skin defect (area, 6×5cm) in a streptozotocin-induced diabetes Wistar rats as a rodent model was used. Group I consisted of diabetic control receiving no MSC; group II, rats received 2 sessions of MSC treatment on day 7 and 10 post-wounding. Group III, rats received 2 sessions of ESWT treatment on day 3 and 7 post-wounding. Each group were assessed at least three wounding-edge skin tissue samples. The proteomic study of skin tissue after different treatment was analyzed. The spots of interest were subjected to in-gel trypsin digestion and MALDI-TOF mass spectrometry in order to elucidate the peptide mass fingerprints. RESULTS: Representative two-dimensional gel electrophoretograms of skin tissue protein in diabetic wound control, post-MSC and post-ESWT treatments. The proteomic study of wounding–edge skin tissue in MSC group had significantly higher abundance of the alpha-2-HS-glycoprotein and vitamin D-binding protein as compared to that in controls. MSC group had significantly lower levels of the Serine protease inhibitor A3N, Serpin B5, tropomyosinα-1 chain, Haptoglobin, as compared to the diabetic controls. In ESWT group had significantly higher abundance of the hemopexin, alpha-2-HS-glycoprotein and vitamin D-binding protein as compared to that in controls. ESWT group had significantly lower levels of the Serine protease inhibitor A3N, Leukocyte elastase inhibitor A, Haptoglobin, Structural maintenance of chromosomes protein 3 and Catechol O-methyltransferase as compared to the diabetic controls without ESWT.Figure 1: Mass spectrometric characteristics of the positive identified proteins in the peri-wounding skin tissue at day 3 post-mesenchymal stem cells treatment.Figure 2: Mass spectrometric characteristics of the positive identified proteins in the peri-wounding skin tissue post- extracorporeal shock wave therapy.CONCLUSION: This proteomic study demonstrated there were some protein indeed up-regulation (alpha-2-HS-glycoprotein and vitamin D-binding protein) or down-regulation (Serine protease inhibitor A3N, Haptoglobin) between MSCs treatment group and ESWT group. Further studies are needed to elucidate whether modulate these proteins could increase diabetic wound healing.

Abstract 4329: Expression of serpin B2, neuroserpin and L1CAM in association with metastasis and survival in non-small cell lung cancer

Abstract Background: Lung cancer is a leading cause of cancer related deaths worldwide, and finding new prognostic and predictive biomarkers are of utmost importance. Previous studies have shown that different serpins and adhesion molecules have an impact on disease progression and development of metastasis, especially to the brain. Here, we examined whether expression of the anti-plasminogen activators Serpin B2 and Neuroserpin, and the adhesion molecule L1CAM was associated with patient prognosis, brain metastasis or other clinicopathologic characteristics, in non-small cell lung cancer (NSCLC). Study design: The material consists of surgical specimens from 100 NSCLC patients treated during 1993-1999 at Haukeland University Hospital, Bergen, Norway. Sections from tumor blocks were stained with antibodies to Serpin B2, Neuroserpin, and L1CAM. Results: There were 66 men and 34 women, with a mean age at diagnosis of 65.7 (41 adenocarcinomas, 38 squamous cell carcinomas, and 21 carcinomas of other types). Observation time ranged from 0 to 254 months (mean 74 months). 12 patients were fully recovered, 46 died of lung cancer, and 34 died from other causes. 11 of 89 patients developed brain metastasis. Low expression of Serpin B2 was associated with reduced overall survival (p<0.05) in adenocarcinomas, and with a trend for all types combined (p = 0.10), whereas there was no significant difference in survival related to expression of Neuroserpin or L1CAM. Expression of Neuroserpin was associated with adrenal metastasis (OR 13.3 (95%CI 1.5-119.7), p<0.05), and there were trends for associations between expression of Serpin B2 and metastases to skin and bone (p = 0.072 and p = 0.068, respectively). There were no significant associations between these markers and presence of brain metastasis. Conclusions: Low expression of Serpin B2 in lung adenocarcinoma was associated with reduced overall survival in our cohort. In contrast to previous findings by others, we did not find a relationship between L1CAM expression and overall survival or metastasis. Neither was there any association between expression of Neuroserpin and survival, although we found an increase in adrenal metastasis in this group. Low expression of Serpin B2 could be a biomarker for prognosis in lung adenocarcinomas and should be studied in other cohorts. Citation Format: Maria Ramnefjell, Lars Helgeland, Lars A. Akslen. Expression of serpin B2, neuroserpin and L1CAM in association with metastasis and survival in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4329. doi:10.1158/1538-7445.AM2015-4329

Abstract 4071: Cancer stem cell-induced vascularization of skin cancer tumors

Abstract Our previous studies identify a subpopulation of epidermal cancer stem (ECS) cells in human squamous cell carcinoma that are efficient tumor formers. These cells display enhanced invasion and migration, and form large, invasive and highly aggressive tumors. We now show that ECS cells produce highly vascularized tumors. This is confirmed visually and by detection of increased expression of the CD31 endothelial cell marker. To understand the molecular basis for increased vascularization, we assayed for ECS cell and non-stem cancer cell production of angiogenic factors. Pro-angiogenic factors, including VEGF-A, HIF-1α and MMP9, are produced at high level in ECS cell-derived tumors, as compared to non-stem cancer cell tumors. In addition, anti-angiogenic factor production (thrombospondin-1, endothelin-1, serpin B5) is reduced. This is consistent with the finding that ECS cell tumor extracts more efficiently stimulate HUVEC cell tube formation. Moreover, these ECS tumor-derived cells display enhanced spheroid formation, and cell migration and invasion. Knockdown of VEGF-A or VEGFR2, or treatment with anti-VEGF-A attenuates these events. In addition, treatment with bevacizumab (anti-VEGF-A) suppresses tumor formation in vivo. These studies suggest that the VEGF-A signaling pathway is enhanced in ECS cells and is in part responsible the ability of these cells to form aggressive and highly vascularized tumors. Citation Format: Dan Grun, Gautam Adhikary, Richard Eckert. Cancer stem cell-induced vascularization of skin cancer tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4071. doi:10.1158/1538-7445.AM2015-4071

Abstract 632: Tumor-targeted fusion constructs containing engineered granzyme B variants with optimized stability and potency

Abstract The serine protease Granzyme B (GrB) is highly cytotoxic generating multiple, intense pro-apoptotic signals when delivered to the cytoplasm of cells and operates through both caspase-dependent and caspase-independent mechanisms. Our laboratory has generated fusion constructs of this payload with VEGF, scFvMEL, BlyS, ML3-9, 4D5 and scFvIT4 targeting molecules thus far. The inhibitor serpin B9 (PI-9) is a 42 kDa protein found at varying levels in cells and plasma binding irreversibly to Granzyme B at the latter's catalytic domain serine (195Serine) resulting in inactive Granzyme B. We undertook modification studies of the GrB primary sequence to improve protein production, serum stability and biological activity. Granzyme B variants were engineered using PCR-based mutagenesis and fused to VEGF121 for direct comparision to the unmodified GrB/VEGF121 fusion protein. Variants designed to prevent binding to PI-9 took into account the impact of the mutation on substrate specificity, and the ability of the mutated GrB to cleave its various cellular substrates. As expected, mutation of 195serine to alanine (S195A) resulted in a complete loss of GrB enzymatic activity as well as in vitro cytotoxicity against VEGFR-1+ and VEGFR-2+ cells. The enzymatic activity of the K27E, R28A (“EA”) double mutant was identical to GrB/VEGF121 when the samples were incubated in PBS. In the presence of 50% human serum, however, the enzymatic activity of GrB/VEGF121 steadily declined to less than 10% over 24 hours while the enzymatic activity of EA-GrB/VEGF121 remained over 40% over that time period. The somewhat similar K27L, R28A (“LA”) double mutant appeared to behave intermediate to the wild-type protein and the EA construct. Both EA and LA GrB mutants retain higher cytotoxic activity after pre-incubation in serum for 4h, compared to unmodified GrB/VEGF121. Mutation of a putative self-cleaving domain and a glycosylation site in GrB appeared to improve enzymatic activity in serum compared to GrB/VEGF121. C210A-GrB/VEGF121, constructed to remove additional thiol sites which may cause aggregation, resulted in a vastly improved yield with otherwise similar serum stability and in vitro cytotoxicity as unmodified GrB/VEGF121. Constructs with modification of the 82PKN84 loop region alone or in combination with the EA mutation had either low activity or had no impact on PI-9 inactivation. Finally, a construct with a caspase-3-cleavable amino acid sequence at the N-terminus showed similar cytotoxic activity to the EA and LA constructs after pre-incubation in serum for 4h. Our studies suggest that these novel GrB variants may significantly improve in vivo half-life. Further studies are underway to determine whether these modifications impact production of soluble material and in vivo therapeutic efficacy. Research conducted, in part, by the Clayton Foundation for Research. Citation Format: Khalid A. Mohamedali, Lawrence H. Cheung, Michael G. Rosenblum. Tumor-targeted fusion constructs containing engineered granzyme B variants with optimized stability and potency. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 632. doi:10.1158/1538-7445.AM2015-632

SERPINB2, TREM2, and DAP12 as biomarkers for leprosy

SERPINB2, TREM2, and DAP12 as biomarkers for leprosy

Association between SNPs in Serpin gene family and risk of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive cancers in the world. Epidemiological survey studies have verified that the development of ESCC relates to a complex interactive process between multiple genetic susceptibilities and environmental exposure. Serpins are a broadly distributed family of protease inhibitors and have been recognized as tumor suppressors in multiple cancer types. While previous studies have reported that Serpin polymorphisms are associated with tumorigenesis, the genetic and functional single nucleotide polymorphisms (SNP) in these genes appear to be complex and remain to be elucidated. In this study, a total of 500 ESCC cases and 500 matched controls in a Southwest China population were evaluated for six SNPs in the exons of three Serpin genes (SerpinB5, SerpinB2, and SerpinE1). Among the six SNPs, the C allele of rs2289519 and rs2289520 in SerpinB5 showed decreased risk of ESCC and the variants might interact with smoking status. Haplotype analysis showed that the T-G haplotype (corresponding to rs2289519-rs2289520) increased the risk of ESCC, while the C-C haplotype decreased the risk. We also found that SerpinB5 gene mRNA expression was significantly downregulated in ESCC cell lines and patient specimen while there is no change in protein structure with different haplotypes. Our results demonstrated that the expression of SerpinB5 was downregulated in ESCC, and the positive SNPs might be associated with a risk of ESCC development.

SERPINB3 (Serpin Peptidase Inhibitor, Clade B (Ovalbumin), Member 3)

Review on SERPINB3, with data on DNA/RNA, on the protein encoded and where the gene is implicated.

Preparation of recombinant serpins B3 and B4 and investigation of their specific interactions with antibodies using hydrogel-based microarrays

The objective of this work was to obtain preparations of recombinant squamous-cell carcinoma antigens (serpins B3 and B4) and to investigate their interactions with different monoclonal antibodies using hydrogel-based microarrays (biochips). Two genetic constructs encoding full-length serpin B3 and serpin B4 molecules were created to produce recombinant SPB3 and SPB4 proteins carrying a N-terminal His6-tag. Monoclonal antibodies against serpin B3 (H3, C5, H5, H81, and G9) were also obtained. An experimental gel-based biological microchip was designed to contain gel elements that carry immobilized antibodies against SPB3, immobilized commercial monoclonal SCC107 and SCC140 antibodies against squamous-cell carcinoma antigen (SCCA), and gel elements with immobilized SPB3 or SPB4. Judging by the specificity of recombinant SPB3 and SPB4, which bind to monoclonal antibodies against SCCA and, according to the manufacturer's data, can recognize conformational epitopes of both SPB3 and SPB4, it was concluded that the obtained recombinant serpins had the correct tertiary structure. A biochip-based direct immunoassay showed that SPB4 could bind effectively only to SCC107 and SCC140 antibodies, while SPB3 interacted specifically not only with these antibodies, but also with H3 and C5 monoclonal antibodies. Using biochip-based sandwich immunoassay, a pair of monoclonal antibodies SCC107/C5 that interacted specifically with serpin B3 but did not interact with serpin B4 was identified. Thus, it has been demonstrated that serpin B3 can be selectively determined in the presence of highly homologous serpin B4 using a biochip-based assay.

Quantification of the placental epigenetic signature of the SERPINB5 gene in maternal plasma of pregnancies complicated by small for gestational age

IntroductionTo investigate the association between pregnancies with small for gestational age (SGA) neonates and the concentration of cell-free fetal DNA or cell-free total DNA in maternal plasma during the first and second trimesters using tissue-specific epigenetic characteristics of the SERPINB5 gene. MethodsA nested case–control study was conducted with maternal plasma collected at 11 to 26 gestational weeks from 51 women with SGA neonates and 102 controls. We performed a real-time quantitative methylation-specific PCR to quantify concentrations of unmethylated-SERPINB5 (U-SERPINB5) as a cell-free fetal DNA marker and methylated-SERPINB5 (M-SERPINB5) as a cell-free total DNA marker. ResultsA positive correlation was observed between U-SERPINB5 and M-SERPINB5 concentrations in both control (r = 0.363, p < 0.001) and SGA groups (r = 0.548, p < 0.001). Moreover, the concentration of U-SERPINB5 or M-SERPINB5 was significantly positive correlated with gestational age at sampling in both controls (U-SERPINB5: r = 0.397, p < 0.001; M-SERPINB5: r = 0.275, p = 0.005) and SGA (U-SERPINB5: r = 0.274, p = 0.052; M-SERPINB5: r = 0.439, p = 0.001). However, the concentration of U-SERPINB5 or M-SERPINB5 was not correlated with birthweight. At 11–14 weeks, U-SERPINB5 and M-SERPINB5 concentrations in SGA did not differ significantly from those of controls. There were also no statistically significant differences in the concentrations of U-SERPINB5 and M-SERPINB5 between SGA and controls at 15–26 weeks of gestation. DiscussionOur findings suggest that U-SERPINB5 and M-SERPINB5 concentrations in maternal plasma during early pregnancy are not associated with pregnancies who delivered SGA neonates.

Proteinase 3 and Serpin B1: a novel pathway in the regulation of caspase-3 activation, neutrophil spontaneous apoptosis, and inflammation.

Neutrophils are the first responders of the inflammatory response. They are characterized by their potent cytotoxic content but also by their limited lifetime. This short half-life is thought to be a self-protecting mechanism for the host, as highlighted by the numerous pathologies associated with imbalanced neutrophil survival. Neutrophil spontaneous death is the prototype of programmed cell death, harboring all the phenotypic hallmarks of apoptosis and dependent on the activation of the effector caspase-3. However, the pathways regulating neutrophil spontaneous death remain ill-defined. In a recent publication, we determined that in aging neutrophils, the cleavage and activation of caspase-3 was mediated by the serine protease Proteinase 3 (PR3), and was independent of the canonical extrinsic and intrinsic apoptosis pathways. In mature neutrophils, PR3 was stored in granules and progressively released to the cytosol during neutrophil aging. The release of PR3 was dependent on lysosomal membrane permeabilization (LMP). Once in the cytosol, PR3 cleaved procaspase-3 at a site upstream of the caspase-9 cleavage site, leading to caspase-3 activation. Inhibition, knockdown or knockout of PR3 delayed neutrophil apoptosis in vitro and in vivo. The adoptive transfer of both WT and PR3-deficient neutrophils to WT mice revealed that the delayed death of neutrophils lacking PR3 in vivo was due to an altered intrinsic apoptosis/survival pathway and not to difference in the inflammatory microenvironment. The cytosolic inhibitor of serine proteases serpin b1 counterbalanced the activity of PR3 in the cytosol of neutrophils, and the deletion of serpinb1 in neutrophils accelerated their spontaneous death. In summary, our results reveal that PR3 and serpinB1 are part of a newly characterized apoptosis pathway, regulating caspase-3 activation and neutrophil spontaneous death and the survival of neutrophils during inflammation.

Interaction of Leukocyte Elastase Inhibitor/L-DNase II with BCL-2 and BAX

Leukocyte Elastase Inhibitor (LEI, also called serpin B1) is a protein involved in apoptosis among other physiological processes. We have previously shown that upon cleavage by its cognate protease, LEI is transformed into L-DNase II, a protein with a pro-apoptotic activity. The caspase independent apoptotic pathway, in which L-DNase II is the final effector, interacts with other pro-apoptotic molecules like Poly-ADP-Ribose polymerase (PARP) or Apoptosis Inducing Factor (AIF). The screening of LEI/L-DNase II interactions showed a possible interaction with several members of the BCL-2 family of proteins which are known to have a central role in the regulation of caspase dependent cell death. In this study, we investigated the regulation of LEI/L-DNase II pathway by two members of this family of proteins: BAX and BCL-2, which have opposite effects on cell survival. We show that, in both BHK and HeLa cells, LEI/L-DNase II can interact with BCL-2 and BAX in apoptotic and non-apoptotic conditions. These proteins which are usually thought to be anti-apoptotic and pro-apoptotic respectively, both inhibit the L-DNase II pro-apoptotic activity. These results give further insight in the regulation of caspase independent pathways and highlight the involvement of the intracellular environment of a given protein in the determinism of its function. They also add a link between caspase-dependent and independent pathways of apoptosis.

Susceptibility to COPD: Differential Proteomic Profiling after Acute Smoking

Cigarette smoking is the main risk factor for COPD (Chronic Obstructive Pulmonary Disease), yet only a subset of smokers develops COPD. Family members of patients with severe early-onset COPD have an increased risk to develop COPD and are therefore defined as “susceptible individuals”. Here we perform unbiased analyses of proteomic profiles to assess how “susceptible individuals” differ from age-matched “non-susceptible individuals” in response to cigarette smoking. Epithelial lining fluid (ELF) was collected at baseline and 24 hours after smoking 3 cigarettes in young individuals susceptible or non-susceptible to develop COPD and older subjects with established COPD. Controls at baseline were older healthy smoking and non-smoking individuals. Five samples per group were pooled and analysed by stable isotope labelling (iTRAQ) in duplicate. Six proteins were selected and validated by ELISA or immunohistochemistry. After smoking, 23 proteins increased or decreased in young susceptible individuals, 7 in young non-susceptible individuals, and 13 in COPD in the first experiment; 23 proteins increased or decreased in young susceptible individuals, 32 in young non-susceptible individuals, and 11 in COPD in the second experiment. SerpinB3 and Uteroglobin decreased after acute smoke exposure in young non-susceptible individuals exclusively, whereas Peroxiredoxin I, S100A9, S100A8, ALDH3A1 (Aldehyde dehydrogenase 3A1) decreased both in young susceptible and non-susceptible individuals, changes being significantly different between groups for Uteroglobin with iTRAQ and for Serpin B3 with iTRAQ and ELISA measures. Peroxiredoxin I, SerpinB3 and ALDH3A1 increased in COPD patients after smoking. We conclude that smoking induces a differential protein response in ELF of susceptible and non-susceptible young individuals, which differs from patients with established COPD. This is the first study applying unbiased proteomic profiling to unravel the underlying mechanisms that induce COPD. Our data suggest that SerpinB3 and Uteroglobin could be interesting proteins in understanding the processes leading to COPD.

Differentially expressed proteins in human breast cancer cells sensitive and resistant to paclitaxel.

The resistance of cancer cells to chemotherapeutic drugs represents a major problem in cancer treatment. Despite all efforts, mechanisms of resistance have not yet been elucidated. To reveal proteins that could be involved in resistance to taxanes, we compared protein expression in whole cell lysates of SK-BR-3 breast cancer cells sensitive to paclitaxel and in lysates of the same line with acquired resistance to paclitaxel. The resistant SK-BR-3 cell line was established in our lab. Protein separation was achieved using high-resolution 2D-electrophoresis, computer analysis and mass spectro-metry. With these techniques we identified four proteins with different expression in resistant SK-BR-3 cells, i.e., serpin B3, serpin B4, heat shock protein 27 (all three upregulated) and cytokeratin 18 (downregulated). Observed changes were confirmed using western blot analysis. This study suggests new directions worthy of further study in the effort to reveal the mechanism of resistance to paclitaxel in breast cancer cells.

Comparative proteome analysis of peripheral neutrophils from sulfur mustard-exposed and COPD patients

Respiratory disorders in sulfur mustard (SM)-exposed and chronic obstructive pulmonary disease (COPD) patients are mostly associated with neutrophilic inflammation, severe airflow limitation, and oxidative stress. The objective of this study was to establish whether neutrophil (PMN) proteomes in these diseases were similar or differed. Blood neutrophil proteomes from healthy, SM-exposed, and COPD subjects were analyzed using two-dimensional gel electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). Elastase activity was determined kinetically. The results showed that levels of S100 calcium-binding protein (CBP) A12, S100 CBP A8, glyceraldehyde-3-phosphate dehy-drogenase, superoxide dismutase, and protein disulfide isomerase proteins – as well as elastase activity – were significantly increased in PMN from ‘diseased’ hosts compared to in cells from healthy controls. In contrast, coactosin-like protein, RhoGDP dissociation inhibitor, and actin isoforms were significantly decreased in diseased subjects’ PMN compared to PMN of healthy controls. Moreover, serpin B1 and coronin-1A were expressed only in PMN of the healthy subjects. Lastly, S100 CBP A9, endoplasmic reticulum (ER)-60 protease, and glutathione-S-transferase isoforms were differentially expressed in the cells from the SM-exposed and COPD subjects. These results show that serpin B1, an efficient inhibitor of neutrophil serine proteases, was not detectable, and elastase activity significantly increased in PMN from both SM-exposed and COPD patients. It seems that, apart from inflammation and oxidative stress, a protease:anti-protease imbalance exists within PMN of both COPD and SM-exposed patients.