Urinary peptidomics analysis reveals proteases involved in diabetic nephropathy

Mechanisms underlying the onset and progression of nephropathy in diabetic patients are not fully elucidated. Deregulation of proteolytic systems is a known path leading to disease manifestation, therefore we hypothesized that proteases aberrantly expressed in diabetic nephropathy (DN) may be involved in the generation of DN-associated peptides in urine. We compared urinary peptide profiles of DN patients (macroalbuminuric, n = 121) to diabetic patients with no evidence of DN (normoalbuminuric, n = 118). 302 sequenced, differentially expressed peptides (adjusted p-value < 0.05) were analysed with the Proteasix tool predicting proteases potentially involved in their generation. Activity change was estimated based on the change in abundance of the investigated peptides. Predictions were correlated with transcriptomics (Nephroseq) and relevant protein expression data from the literature. This analysis yielded seventeen proteases, including multiple forms of MMPs, cathepsin D and K, kallikrein 4 and proprotein convertases. The activity of MMP-2 and MMP-9, predicted to be decreased in DN, was investigated using zymography in a DN mouse model confirming the predictions. Collectively, this proof-of-concept study links urine peptidomics to molecular changes at the tissue level, building hypotheses for further investigation in DN and providing a workflow with potential applications to other diseases

Comparative study of secreted proteins from cervical normal and cancer cell lines for the discovery of cancer biomarkers

Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, as well as putative biomarkers, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed via 2 proteomic methods 1) 2DE-MALDI TOF-MS and 2) LC-MS/MS. Proteomics analysis revealed new information (protein targets) for the molecular characterization of cervical cancer (e.g. beta ig-h3, PRDX2, SOD2, FUCA1, ADAM10, CATD). Bioinformatics tools were employed for the prediction of deregulated pathways, in which secreted proteins would participate (NRF2-mediated oxidative stress response, Inhibition of matrix metalloproteases). The results of bioinformatics analysis as well as further protein-targets were validated via immunoassays, enzymatic methods as well as with the method of high sensitivity MRM (eg. ΤΙΜP1, CATD etc). In conclusion, the secreted proteins identified in cervical cancer cell lines propose putative biomarkers and potential therapeutic targets.Κατά την καρκινογένεση, τα καρκινικά κύτταρα παράγουν εκκριτώματα με διαφορετικά ποιοτικά και ποσοτικά χαρακτηριστικά. Σκοπός της εργασίας ήταν η ανακάλυψη βιολογικών διαδικασιών που εμπλέκονται στην καρκινογένεση του τραχήλου της μήτρας, και πιθανών βιοδεικτών με την πρωτεωμική ανάλυση τεσσάρων αντιπροσωπευτικών κυτταρικών σειρών του τραχήλου της μήτρας: τη SiHa (HPV16+), τη HeLa (HPV18+), τη C33A (HPV-), και την HCK1T (φυσιολογική). Xρησιμοποιήθηκαν 2 πρωτεωμικές προσεγγίσεις για την επίτευξη του παραπάνω στόχου 1) Δισδιάστατη ηλεκτροφόρηση συζευγμένη με φασματομετρία μάζας (2DE-MALDI TOF-MS) και 2) Υγρή χρωματογραφία συζευγμένη με φασματομετρία μάζας (LC-MS/MS). Η πρωτεωμική ανάλυση απέδωσε νέες πληροφορίες (πρωτεΐνες-στόχοι) για τον μοριακό χαρακτηρισμό του καρκίνου του τραχήλου της μήτρας (π.χ beta ig-h3, PRDX2, SOD2, FUCA1, ADAM10, CATD). Ακολούθησε μια ολοκληρωμένη βιοπληροφορική ανάλυση και για τις 2 μεθοδολογίες η οποία απέδωσε απορρυθμισμένα βιολογικά μονοπάτια (π.χ. «Επαγωγή αντιοξειδωτικών πρωτεϊνών μέσω του μεταγραφικού παράγοντα NRF2» και «Καταστολή μεταλλοπρωτεϊνασών»). Τα αποτελέσματα της βιοπληροφορικής ανάλυσης καθώς και περαιτέρω πρωτεΐνες-στόχοι επιβεβαιώθηκαν με ανοσολογικές, ενζυμικές μεθόδους καθώς και τη μέθοδο υψηλής ευαισθησίας MRM (πχ ΤΙΜP1, CATD κτλ). Συμπερασματικά, αναγνωρίστηκαν εκκρινόμενες πρωτεΐνες οι οποίες μπορεί να είναι πιθανοί βιοδείκτες ή θεραπευτικοί στόχοι

Συγκριτική μελέτη εκκρινόμενων πρωτεϊνών από μια φυσιολογική και καρκινικές κυτταρικές σειρές του τραχήλου της μήτρας για την ανάδειξη καρκινικών βιοδεικτών

Κατά την καρκινογένεση, τα καρκινικά κύτταρα παράγουν εκκριτώματα με διαφορετικά ποιοτικά και ποσοτικά χαρακτηριστικά. Σκοπός της εργασίας ήταν η ανακάλυψη βιολογικών διαδικασιών που εμπλέκονται στην καρκινογένεση του τραχήλου της μήτρας, και πιθανών βιοδεικτών με την πρωτεωμική ανάλυση τεσσάρων αντιπροσωπευτικών κυτταρικών σειρών του τραχήλου της μήτρας: τη SiHa (HPV16+), τη HeLa (HPV18+), τη C33A (HPV-), και την HCK1T (φυσιολογική). Xρησιμοποιήθηκαν 2 πρωτεωμικές προσεγγίσεις για την επίτευξη του παραπάνω στόχου 1) Δισδιάστατη ηλεκτροφόρηση συζευγμένη με φασματομετρία μάζας (2DE-MALDI TOF-MS) και 2) Υγρή χρωματογραφία συζευγμένη με φασματομετρία μάζας (LC-MS/MS). Η πρωτεωμική ανάλυση απέδωσε νέες πληροφορίες (πρωτεΐνες-στόχοι) για τον μοριακό χαρακτηρισμό του καρκίνου του τραχήλου της μήτρας (π.χ beta ig-h3, PRDX2, SOD2, FUCA1, ADAM10, CATD). Ακολούθησε μια ολοκληρωμένη βιοπληροφορική ανάλυση και για τις 2 μεθοδολογίες η οποία απέδωσε απορρυθμισμένα βιολογικά μονοπάτια (π.χ. «Επαγωγή αντιοξειδωτικών πρωτεϊνών μέσω του μεταγραφικού παράγοντα NRF2» και «Καταστολή μεταλλοπρωτεϊνασών»). Τα αποτελέσματα της βιοπληροφορικής ανάλυσης καθώς και περαιτέρω πρωτεΐνες-στόχοι επιβεβαιώθηκαν με ανοσολογικές, ενζυμικές μεθόδους καθώς και τη μέθοδο υψηλής ευαισθησίας MRM (πχ ΤΙΜP1, CATD κτλ). Συμπερασματικά, αναγνωρίστηκαν εκκρινόμενες πρωτεΐνες οι οποίες μπορεί να είναι πιθανοί βιοδείκτες ή θεραπευτικοί στόχοι.Cancer cells acquire unique secretome compositions that contribute to tumor development and metastasis. The aim of our study was to elucidate the biological processes involved in cervical cancer, as well as putative biomarkers, by performing a proteomic analysis of the secretome from the following informative cervical cell lines: SiHa (HPV16+), HeLa (HPV18+), C33A (HPV−), and HCK1T (normal). Proteins were analyzed via 2 proteomic methods 1) 2DE-MALDI TOF-MS and 2) LC-MS/MS. Proteomics analysis revealed new information (protein targets) for the molecular characterization of cervical cancer (e.g. beta ig-h3, PRDX2, SOD2, FUCA1, ADAM10, CATD). Bioinformatics tools were employed for the prediction of deregulated pathways, in which secreted proteins would participate (NRF2-mediated oxidative stress response, Inhibition of matrix metalloproteases). The results of bioinformatics analysis as well as further protein-targets were validated via immunoassays, enzymatic methods as well as with the method of high sensitivity MRM (eg. ΤΙΜP1, CATD etc). In conclusion, the secreted proteins identified in cervical cancer cell lines propose putative biomarkers and potential therapeutic targets

Novel structural approaches concerning HPV proteins: Insight into targeted therapies for cervical cancer (Review)

Cervical cancer incidence is tightly linked to HPV infection, and particularly virus types 16 and 18 cause the majority of cases presenting with pre-cancerous stages of cervical intraepithelial neoplasia (CIN). Structural and functional information concerning HPV proteins can offer novel insight into the mechanism(s) of cancer progression in the cervical epithelium. Recently, novel structural determinants of the interactions of viral proteins with their targets in keratinocytes have been elucidated. These exciting findings open the way for the development of targeted anti-oncogenic therapies, and may eventually allow the introduction of novel approaches for a rational cervical cancer treatment

Proteomics approaches in cervical cancer: focus on the discovery of biomarkers for diagnosis and drug treatment monitoring

Introduction: The HPV virus accounts for the majority of cervical cancer cases. Although a diagnostic tool (Pap Test) is widely available, cervical cancer incidence still remains high worldwide, and especially in developing countries, attributed to a large extent to suboptimal sensitivities of the Pap test and unavailability of the test in developing countries.Areas covered: Proteomics approaches have been used in order to understand the HPV virus correlation to cervical cancer pathology, as well as to discover putative biomarkers for early cervical cancer diagnosis and drug mode of action.Expert commentary: The present review summarizes the latest in vitro and in vivo proteomic studies for the discovery of putative cervical cancer biomarkers and the evaluation of available drugs and treatments

In Situ Peroxidase Labeling Followed by Mass-Spectrometry Reveals TIA1 Interactome

TIA1 is a broadly expressed DNA/RNA binding protein that regulates multiple aspects of RNA metabolism. It is best known for its role in stress granule assembly during the cellular stress response. Three RNA recognition motifs mediate TIA1 functions along with a prion-like domain that supports multivalent protein-protein interactions that are yet poorly characterized. Here, by fusing the enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to TIA1 combined with mass spectrometry, the proteins in the immediate vicinity of TIA1 were defined in situ. Eighty-six and 203 protein partners, mostly associated with ribonucleoprotein complexes, were identified in unstressed control and acute stress conditions, respectively. Remarkably, the repertoire of TIA1 protein partners was highly dissimilar between the two cellular states. Under unstressed control conditions, the biological processes associated with the TIA1 interactome were enriched for cytosolic ontologies related to mRNA metabolism, such as translation initiation, nucleocytoplasmic transport, and RNA catabolism, while the protein identities were primarily represented by RNA binding proteins, ribosomal subunits, and eicosanoid regulators. Under acute stress, TIA1-labeled partners displayed a broader subcellular distribution that included the chromosomes and mitochondria. The enriched biological processes included splicing, translation, and protein synthesis regulation, while the molecular function of the proteins was enriched for RNA binding activity, ribosomal subunits, DNA double-strand break repair, and amide metabolism. Altogether, these data highlight the TIA1 spatial environment with its different partners in diverse cellular states and pave the way to dissect TIA1 role in these processes

APEX2-Mediated Proximity Labeling Resolves the DDIT4-Interacting Proteome

DNA damage-inducible transcript 4 (DDIT4) is a ubiquitous protein whose expression is transiently increased in response to various stressors. Chronic expression has been linked to various pathologies, including neurodegeneration, inflammation, and cancer. DDIT4 is best recognized for repressing mTORC1, an essential protein complex activated by nutrients and hormones. Accordingly, DDIT4 regulates metabolism, oxidative stress, hypoxic survival, and apoptosis. Despite these well-defined biological functions, little is known about its interacting partners and their unique molecular functions. Here, fusing an enhanced ascorbate peroxidase 2 (APEX2) biotin-labeling enzyme to DDIT4 combined with mass spectrometry, the proteins in the immediate vicinity of DDIT4 in either unstressed or acute stress conditions were identified in situ. The context-dependent interacting proteomes were quantitatively but not functionally distinct. DDIT4 had twice the number of interaction partners during acute stress compared to unstressed conditions, and while the two protein lists had minimal overlap in terms of identity, the proteins’ molecular function and classification were essentially identical. Moonlighting keratins and ribosomal proteins dominated the proteomes in both unstressed and stressed conditions, with many of their members having established non-canonical and indispensable roles during stress. Multiple keratins regulate mTORC1 signaling via the recruitment of 14-3-3 proteins, whereas ribosomal proteins control translation, cell cycle progression, DNA repair, and death by sequestering critical proteins. In summary, two potentially distinct mechanisms of DDIT4 molecular function have been identified, paving the way for additional research to confirm and consolidate these findings

Membrane proteomics of cervical cancer cell lines reveal insights on the process of cervical carcinogenesis

The available therapeutic approaches for cervical cancer can seriously affect the fertility potential of patient; thus, there is a pressing requirement for less toxic and targeted therapies. The membrane proteome is a potential source of therapeutic targets; however, despite the significance of membrane proteins in cancer, proteomic analysis has been a challenging task due to their unique biochemical properties. The aim of the present study was to develop an efficient membrane protein enrichment protocol, and to the best of our knowledge, to compare for the first time the expression pattern of membrane proteins of one normal cell line, HCK1T, and three cervical cancer cell lines, C33A, a human papilloma virus (HPV)-negative cell line, and two HPV-positive cell lines, SiHa (HPV16(+)) and HeLa (HPV18(+)). The study aimed to identify the proteins that are involved in cervical carcinogenesis and may constitute novel drug targets. Membrane protein isolation, liquid chromatography coupled with tandem mass spectrometry proteomics and bioinformatics analysis were performed in the membrane fraction of the informative cervical cell lines following a novel enrichment protocol. The percentages of membrane and transmembrane proteins in the enrichment protocol were higher compared with those of the corresponding data derived from total cell extract analysis. Differentially expressed proteins were detected by the comparison of the cervical cancer cell lines with the normal cell line. These proteins constitute molecular features of cancer pathology and participate in biological pathways relevant to malignancy, including HIPPO signaling', PI3K/Akt signaling', cell cycle: G2/M DNA damage checkpoint regulation' and EIF2 signaling'. These unique membrane protein identifications offer insights on a previously inaccessible region of the cervical cancer proteome, and may represent putative diagnostic and prognostic markers, and eventually therapeutic targets

Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation

Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudo-typed lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6 x 10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy. (C) 2013 Elsevier B.V. All rights reserved