Basic and clinical applications of proteomic research. Our experience

In this thesis we applied proteomic techniques to basic biological topics as well as to clinically relevant issues both in diagnostic and in investigative perspectives. The procedure we employed consisted of electrophoretic separation of the relevant proteins, in-gel-proteolytic fragmentation and finally identification either of precise sites of labelling in irreversible protein PTM by Tgase 2 or of the protein itself in clinically oriented oncologic studies. In protein PTM we have analysed three substrate proteins, troponin, tubulin and mutant dyphtheria toxin. We obtained evidence that TnT was the subunit modified in native troponin and that glutamine 13 is the site of Q-labelling, while the protein is not a K substrate. Data on tubulin and DT are not yet so advanced. Both proteins are however substrates of Tgase 2. Tubulin is better substrate when in the aggregated form and is labelled in the C-terminal region. Moderate crosslinking also occurred. The protein when we focused on is the DT mutant CRM197, which instead undergoes massive crosslinkage forming protein aggregates whose accumulatioon is competed by incorporation of external amines, confirming the occurrence of g-glutamyl-e-lysine isopeptide bonds. In the oncologic studies we used the bi-dimensional electrophoresis and mass spectrometry approach to isolate specific proteins and we identified from breast cancer tissues to detect candidate disease biomarkers for diagnosis and prognosis and proteins involved in Epithelial Mesenchymal Transition in cholangiocarcinoma cell cells. In our four investigations we took particularly of the technical aspects to optimize resolution of the experimental data in both perspectives

Potential role of circulating microRNAs as early markers of preeclampsia

Abstract Objective To identify microRNAs (miRNAs) differentially expressed at early stages of gestation (12–14 weeks) in the serum of pregnant women, who later developed severe preeclampsia (sPE) in the third trimester of pregnancy ( n = 24) compared to women with normal pregnancy ( n = 24). Materials and Methods Sera from 12–14-week-gestation whole blood were subjected to microarray analysis with TaqMan Low Density Array chips (human microRNA panel V3.0), and to quantitative real-time polymerase chain reaction. Results By using the TaqMan Low Density Array chip technology, 19 mature miRNAs appeared differentially expressed in the group of women who later developed sPE as compared to normal women. The expression of four miRNAs (miR-1233, miR-520, miR-210, miR-144) was validated by quantitative real-time polymerase chain reaction analysis. MiR-1233 was the most overexpressed in the serum of women who later developed sPE. Conclusion Circulating miRNAs deserve further investigation in order to explore their potential role in the pathogenesis of preeclampsia. In particular, miR-1233 might represent a potential marker of early sPE

Dysregulated chaperones associated with cell proliferation and negative apoptosis regulation in the uterine leiomyoma

Uterine leiomyomas are benign smooth muscle cell tumors that originate from the myometrium. In this study we focus on dysregulated chaperones associated with cell proliferation and apoptosis. Paired tissue samples of 15 leiomyomas and adjacent myometria were obtained and analyzed by two-dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification and western blotting for 2-DE data validation. The values of 6 chaperones were found to be significantly different in the leiomyoma when compared with the myometrium. A total of 4 proteins were upregulated in the leiomyoma and 2 proteins were downregulated. Calreticulin and 78 kDa glucose-regulated protein were further validated by western blotting because the first is considered a marker of cell proliferation, while the second protects against apoptotic cell death. In addition, we also validated the two downregulated proteins heat shock protein \u3b2-1 and heat shock 70 kDa protein 1A. Our study shows the existence of a dysregulation of chaperone proteins associated with leiomyoma development. Functional studies are needed to ascertain the role of these chaperones in the leiomyoma. This may be crucial for the further development of specific inhibitors against the activity of these proteins in order to block the growth of the leiomyoma

A Targeted Proteomics Approach for Screening Serum Biomarkers Observed in the Early Stage of Type I Endometrial Cancer

Endometrial cancer (EC) is the most common gynecologic malignancy, and it arises in the inner part of the uterus. Identification of serum biomarkers is essential for diagnosing the disease at an early stage. In this study, we selected 44 healthy controls and 44 type I EC at tumor stage 1, and we used the Immuno-oncology panel and the Target 96 Oncology III panel to simultaneously detect the levels of 92 cancer-related proteins in serum, using a proximity extension assay. By applying this methodology, we identified 20 proteins, associated with the outcome at binary logistic regression, with a p-value below 0.01 for the first panel and 24 proteins with a p-value below 0.02 for the second one. The final multivariate logistic regression model, combining proteins from the two panels, generated a model with a sensitivity of 97.67% and a specificity of 83.72%. These results support the use of the proposed algorithm after a validation phase

Changes in protein expression in two cholangiocarcinoma cell lines undergoing formation of multicellular tumor spheroids In vitro

Epithelial-to-Mesenchymal Transition (EMT) is relevant in malignant growth and frequently correlates with worsening disease progression due to its implications in metastases and re- sistance to therapeutic interventions. Although EMT is known to occur in several types of solid tumors, the information concerning tumors arising from the epithelia of the bile tract is still limited. In order to approach the problem of EMT in cholangiocarcinoma, we decided to investigate the changes in protein expression occurring in two cell lines under conditions leading to growth as adherent monolayers or to formation of multicellular tumor spheroids (MCTS), which are considered culture models that better mimic the growth characteristics of in-vivo solid tumors. In our system, changes in phenotypes occur with only a decrease in transmembrane E-cadherin and vimentin expression, minor changes in the transglutami- nase protein/activity but with significant differences in the proteome profiles, with declining and increasing expression in 6 and in 16 proteins identified by mass spectrometry. The aris- ing protein patterns were analyzed based on canonical pathways and network analysis. These results suggest that significant metabolic rearrangements occur during the conver- sion of cholangiocarcinomas cells to the MCTS phenotype, which most likely affect the car- bohydrate metabolism, protein folding, cytoskeletal activity, and tissue sensitivity to oxygen

Proteomic study identifies glycolytic and inflammation pathways involved in recurrent otitis media

Recurrent acute otitis media (RAOM) in children is clinically defined as the occurrence of at least three episodes of acute otitis media over a course of 6 months. A further common pathological condition of interest in the context of pediatric otolaryngology is adenotonsillar hypertrophy (ATH), a common cause of obstructive sleep apnea syndrome. Aimed at unraveling the differential modulation of proteins in the two pathologies and at understanding the possible pathways involved in their onset, we analyzed the proteomic profile of the adenoids from 14 RAOM and ATH patients by using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The 2-DE coupled with MS allowed us to identify 23 spots with significant (p-value < 0.05) changes in protein amount, recognizing proteins involved in neutrophil degranulation and glycolysis pathways.This research was funded by I.R.C.C.S. “B. G.”, grant number RC 02/20 and RC 15/17

A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach

Association between up-regulated expression proteins and circulating steroidal hormones in leiomyoma

Several studies have shown that both ovarian hormones estrogen and progesterone and various proteins are involved in uterine leiomyoma growth. We hereby describe a study carried out to investigate the possible relation between progesterone, estrone, 17\u3b2-estradiol and the seven dysregulated proteins in the leiomyoma. Statistical analyses showed a significant inverse rank correlation between desmin expressed in leiomyoma and progesterone (\u3c1 = 120.9276; p = 0.0077) and between alpha-1-antitrypsin expressed in leiomyoma and 17\u3b2-estradiol (\u3c1 = 120.8571; p = 0.0137). Our data suggest that decreased levels of 17-\u3b2-estradiol involve an increasing of the inflammation response stimulating alpha-1-antitrypsin expression in leiomyoma and that lower levels of progesterone associated with increasing of desmin expression may be related to increase of inflammation response, and to the role played by desmin in signal transduction inducing leiomyoma growth

Abnormal expression of leiomyoma cytoskeletal proteins involved in cell migration

Uterine leiomyomas are monoclonal tumors. Several factors are involved in the neoplastic transformation of the myometrium. In our study we focused on dysregulated cytoskeletal proteins in the leiomyoma as compared to the myometrium. Paired tissue samples of ten leiomyomas and adjacent myometria were obtained and analyzed by two\u2011dimensional gel electrophoresis (2-DE). Mass spectrometry was used for protein identification, and western blotting for 2-DE data validation. The values of ten cytoskeletal proteins were found to be significantly different: eight proteins were upregulated in the leiomyoma and two proteins were downregulated. Three of the upregulated proteins (myosin regulatory light polypeptide 9, four and a half LIM domains protein 1 and LIM and SH3 domain protein 1) are involved in cell migration, while downregulated protein transgelin is involved in replicative senescence. Myosin regulatory light polypeptide 9 (MYL9) was further validated by western blotting because it is considered to be a cell migration marker in several cancers and could play a key role in leiomyoma development. Our data demonstrate significant alterations in the expression of cytoskeletal proteins involved in leiomyoma growth. A better understanding of the involvement of cytoskeletal proteins in leiomyoma pathogenesis may contribute to the identification of new therapeutic targets and the development of new pharmacological approaches

A Proteomic Approach for the Identification of Up-Regulated Proteins Involved in the Metabolic Process of the Leiomyoma

Uterine leiomyoma is the most common benign smooth muscle cell tumor of the uterus. Proteomics is a powerful tool for the analysis of complex mixtures of proteins. In our study, we focused on proteins that were upregulated in the leiomyoma compared to the myometrium. Paired samples of eight leiomyomas and adjacent myometrium were obtained and submitted to two-dimensional gel electrophoresis (2-DE) and mass spectrometry for protein identification and to Western blotting for 2-DE data validation. The comparison between the patterns revealed 24 significantly upregulated (p &lt; 0.05) protein spots, 12 of which were found to be associated with the metabolic processes of the leiomyoma and not with the normal myometrium. The overexpression of seven proteins involved in the metabolic processes of the leiomyoma was further validated by Western blotting and 2D Western blotting. Four of these proteins have never been associated with the leiomyoma before. The 2-DE approach coupled with mass spectrometry, which is among the methods of choice for comparative proteomic studies, identified a number of proteins overexpressed in the leiomyoma and involved in several biological processes, including metabolic processes. A better understanding of the mechanism underlying the overexpression of these proteins may be important for therapeutic purposes