Biochemical Characterisation of Human Transglutaminase 4

Transglutaminases are protein-modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is involved in the development of autoimmune and tumour diseases. Although rodent TG4 is well characterised, biochemical characteristics of human TG4 that could help th e understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtitre plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell–cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

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Biochemical characterisation of human transglutaminase 4

10.1101/2021.09.01.458359 ◽

2021 ◽

Author(s):

Zsuzsa Csobán-Szabó ◽

Bálint Bécsi ◽

Saïd El Alaoui ◽

László Fésüs ◽

Ilma Rita Korponay-Szabó ◽

...

Keyword(s):

Cell Interaction ◽

Microtiter Plate ◽

Cell Extract ◽

Proteolytic Activation ◽

293 Cells ◽

Biochemical Characterisation ◽

Cancer Invasiveness ◽

Reducing Environment ◽

Regulatory Event

AbstractTransglutaminases are protein modifying enzymes involved in physiological and pathological processes with potent therapeutic possibilities. Human TG4, also called prostate transglutaminase, is frequently associated with pathological symptoms and particularly with cancer invasiveness. Although rodent TG4 is well characterised, bio-chemical characteristics of human TG4 that could help the understanding of its way of action are not published. First, we analysed proteomics databases and found that TG4 protein is present in human tissues beyond the prostate. Then, we studied in vitro the transamidase activity of human TG4 and its regulation using the microtiter plate method. Human TG4 has low transamidase activity which prefers slightly acidic pH and a reducing environment. It is enhanced by submicellar concentrations of SDS suggesting that membrane proximity is an important regulatory event. Human TG4 does not bind GTP as tested by GTP-agarose and BODIPY-FL-GTPγS binding, and its proteolytic activation by dispase or when expressed in AD-293 cells was not observed either. We identified several potential human TG4 glutamine donor substrates in the AD-293 cell extract by biotin-pentylamine incorporation and mass spectrometry. Several of these potential substrates are involved in cell-cell interaction, adhesion and proliferation, suggesting that human TG4 could become an anticancer therapeutic target.

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Preliminary Study on Pasta Samples Characterized in Antioxidant Compounds and Their Biological Activity on Kidney Cells

Nutrients ◽

10.3390/nu13041131 ◽

2021 ◽

Vol 13 (4) ◽

pp. 1131 ◽

Cited By ~ 1

Author(s):

Federico Di Marco ◽

Francesco Trevisani ◽

Pamela Vignolini ◽

Silvia Urciuoli ◽

Andrea Salonia ◽

...

Keyword(s):

Indoleacetic Acid ◽

Antioxidant Properties ◽

Antioxidant Compounds ◽

Kidney Cells ◽

Hek 293 ◽

293 Cells ◽

High Concentrations ◽

Β Carotene ◽

Egg Pasta

Pasta is one of the basic foods of the Mediterranean diet and for this reason it was chosen for this study to evaluate its antioxidant properties. Three types of pasta were selected: buckwheat, rye and egg pasta. Qualitative–quantitative characterization analyses were carried out by HPLC-DAD to identify antioxidant compounds. The data showed the presence of carotenoids such as lutein and polyphenols such as indoleacetic acid, (carotenoids from 0.08 to 0.16 mg/100 g, polyphenols from 3.7 to 7.4 mg/100 g). To assess the effect of the detected metabolites, in vitro experimentation was carried out on kidney cells models: HEK-293 and MDCK. Standards of β-carotene, indoleacetic acid and caffeic acid, hydroalcoholic and carotenoid-enriched extracts from samples of pasta were tested in presence of antioxidant agent to determine viability variations. β-carotene and indoleacetic acid standards exerted a protective effect on HEK-293 cells while no effect was detected on MDCK. The concentrations tested are likely in the range of those reached in body after the consumption of a standard pasta meal. Carotenoid-enriched extracts and hydroalcoholic extracts showed different effects, observing rescues for rye pasta hydroalcoholic extract and buckwheat pasta carotenoid-enriched extract, while egg pasta showed milder dose depending effects assuming pro-oxidant behavior at high concentrations. The preliminary results suggest behaviors to be traced back to the whole phytocomplexes respect to single molecules and need further investigations.

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Inhibition of human coagulation factor VIII by monoclonal antibodies. Mapping of functional epitopes with the use of recombinant factor VIII fragments

Biochemical Journal ◽

10.1042/bj2630187 ◽

1989 ◽

Vol 263 (1) ◽

pp. 187-194 ◽

Cited By ~ 41

Author(s):

A Leyte ◽

K Mertens ◽

B Distel ◽

R F Evers ◽

M J M De Keyzer-Nellen ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Factor Viii ◽

Factor Xa ◽

Coagulation Factor ◽

Procoagulant Activity ◽

Coagulation Factor Viii ◽

Coagulation Cascade ◽

Restriction Enzyme Digestion ◽

Proteolytic Activation

The epitopes of four monoclonal antibodies against coagulation Factor VIII were mapped with the use of recombinant DNA techniques. Full-length Factor VIII cDNA and parts thereof were inserted into the vector pSP64, permitting transcription in vitro with the use of a promoter specific for SP6 RNA polymerase. Factor VIII DNA inserts were truncated from their 3′-ends by selective restriction-enzyme digestion and used as templates for ‘run-off’ mRNA synthesis. Translation in vitro with rabbit reticulocyte lysate provided defined radiolabelled Factor VIII fragments for immunoprecipitation studies. Two antibodies are shown to be directed against epitopes on the 90 kDa chain of Factor VIII, between residues 712 and 741. The 80 kDa chain appeared to contain the epitopes of the other two antibodies, within the sequences 1649-1778 and 1779-1840 respectively. The effect of antibody binding to these sequences was evaluated at two distinct levels within the coagulation cascade. Both Factor VIII procoagulant activity and Factor VIII cofactor function in Factor Xa generation were neutralized upon binding to the region 1779-1840. The antibodies recognizing the region 713-740 or 1649-1778, though interfering with Factor VIII procoagulant activity, did not inhibit in Factor Xa generation. These findings demonstrate that antibodies that virtually inhibit Factor VIII in coagulation in vitro are not necessarily directed against epitopes involved in Factor VIII cofactor function. Inhibition of procoagulant activity rather than of cofactor function itself may be explained by interference in proteolytic activation of Factor VIII. This hypothesis is in agreement with the localization of the epitopes in the proximity of thrombin-cleavage or Factor Xa-cleavage sites.

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The 5- flanking sequences of Drosophila tRNAArg genes control their in vitro transcription in a Drosophila cell extract.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)33342-8 ◽

1982 ◽

Vol 257 (24) ◽

pp. 14738-14744 ◽

Cited By ~ 13

Author(s):

T Dingermann ◽

D J Burke ◽

S Sharp ◽

J Schaack ◽

D Söll

Keyword(s):

In Vitro Transcription ◽

Cell Extract ◽

Drosophila Cell ◽

Flanking Sequences

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ATRT-14. MACROPHAGE-TUMOR CELL INTERACTION PROMOTES ATRT PROGRESSION AND CHEMORESISTANCE

Neuro-Oncology ◽

10.1093/neuonc/noaa222.013 ◽

2020 ◽

Vol 22 (Supplement_3) ◽

pp. iii278-iii278

Author(s):

Viktoria Melcher ◽

Monika Graf ◽

Marta Interlandi ◽

Natalia Moreno ◽

Flavia W de Faria ◽

...

Keyword(s):

Tumor Cell ◽

Tumor Cells ◽

Chemotherapy Resistance ◽

Cell Communication ◽

Xenograft Model ◽

Cell Interaction ◽

Immunofluorescent Staining ◽

Genetic Traits ◽

Rhabdoid Tumors

Abstract Atypical teratoid/rhabdoid tumors (ATRT) are pediatric brain neoplasms that are known for their heterogeneity concerning pathophysiology and outcome. The three genetically rather uniform but epigenetically distinct molecular subgroups of ATRT alone do not sufficiently explain the clinical heterogeneity. Therefore, we examined the tumor microenvironment (TME) in the context of tumor diversity. By using multiplex-immunofluorescent staining and single-cell RNA sequencing (scRNA-seq) we unveiled the pan-macrophage marker CD68 as a subgroup-independent negative prognostic marker for survival of ATRT patients. ScRNA-seq analysis of murine ATRT-SHH, ATRT-MYC and extracranial RT (eRT) provide a delineation of the TME, which is predominantly infiltrated by myeloid cells: more specifically a microglia-enriched niche in ATRT-SHH and a bone marrow-derived macrophage infiltration in ATRT-MYC and eRT. Exploring the cell-cell communication of tumor cells with tumor-associated immune cells, we found that Cd68+ tumor-associated macrophages (TAMs) are central to intercellular communication with tumor cells. Moreover, we uncovered distinct tumor phenotypes in murine ATRT-MYC that share genetic traits with TAMs. These intermediary cells considerably increase the intratumoral heterogeneity of ATRT-MYC tumors. In vitro co-culture experiments recapitulated the capability of ATRT-MYC cells to interchange cell material with macrophages extensively, in contrast to ATRT-SHH cells. We found that microglia are less involved in the exchange of information with ATRT cells and that direct contact is a prerequisite for incorporation. A relapse xenograft model implied that intermediary cells are involved in the acquisition of chemotherapy resistance. We show evidence that TAM-tumor cell interaction is one mechanism of chemotherapy resistance and relapse in ATRT.

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Furin Processing and Proteolytic Activation of Semliki Forest Virus

Journal of Virology ◽

10.1128/jvi.77.5.2981-2989.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 2981-2989 ◽

Cited By ~ 65

Author(s):

Xinyong Zhang ◽

Martin Fugère ◽

Robert Day ◽

Margaret Kielian

Keyword(s):

Conformational Changes ◽

Secretory Pathway ◽

Semliki Forest Virus ◽

Fusion Reaction ◽

Low Ph ◽

Protein Fusion ◽

Proteolytic Activation ◽

Control Virus

ABSTRACT The alphavirus Semliki Forest virus (SFV) infects cells via a low-pH-dependent membrane fusion reaction mediated by the E1 envelope protein. Fusion is regulated by the interaction of E1 with the receptor-binding protein E2. E2 is synthesized as a precursor termed “p62,” which forms a stable heterodimer with E1 and is processed late in the secretory pathway by a cellular furin-like protease. Once processing to E2 occurs, the E1/E2 heterodimer is destabilized so that it is more readily dissociated by exposure to low pH, allowing fusion and infection. We have used FD11 cells, a furin-deficient CHO cell line, to characterize the processing of p62 and its role in the control of virus fusion and infection. p62 was not cleaved in FD11 cells and cleavage was restored in FD11 cell transfectants expressing human furin. Studies of unprocessed virus produced in FD11 cells (wt/p62) demonstrated that the p62 protein was efficiently cleaved by purified furin in vitro, without requiring prior exposure to low pH. wt/p62 virus particles were also processed during their endocytic uptake in furin-containing cells, resulting in more efficient virus infection. wt/p62 virus was compared with mutant L, in which p62 cleavage was blocked by mutation of the furin-recognition motif. wt/p62 and mutant L had similar fusion properties, requiring a much lower pH than control virus to trigger fusion and fusogenic E1 conformational changes. However, the in vivo infectivity of mutant L was more strongly inhibited than that of wt/p62, due to additional effects of the mutation on virus-cell binding.

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SUMOylation Negatively Regulates Angiogenesis by Targeting Endothelial NOTCH Signaling

Circulation Research ◽

10.1161/circresaha.117.310696 ◽

2017 ◽

Vol 121 (6) ◽

pp. 636-649 ◽

Cited By ~ 15

Author(s):

Xiaolong Zhu ◽

Sha Ding ◽

Cong Qiu ◽

Yanna Shi ◽

Lin Song ◽

...

Keyword(s):

Notch Signaling ◽

Cell Fate ◽

Developmental Disorders ◽

Interaction Mechanism ◽

Growth Factor Receptor ◽

Vascular Diseases ◽

Cell Interaction ◽

Protein Signaling

Rationale: The highly conserved NOTCH (neurogenic locus notch homolog protein) signaling pathway functions as a key cell–cell interaction mechanism controlling cell fate and tissue patterning, whereas its dysregulation is implicated in a variety of developmental disorders and cancers. The pivotal role of endothelial NOTCH in regulation of angiogenesis is widely appreciated; however, little is known about what controls its signal transduction. Our previous study indicated the potential role of post-translational SUMO (small ubiquitin-like modifier) modification (SUMOylation) in vascular disorders. Objective: The aim of this study was to investigate the role of SUMOylation in endothelial NOTCH signaling and angiogenesis. Methods and Results: Endothelial SENP1 (sentrin-specific protease 1) deletion, in newly generated endothelial SENP1 (the major protease of the SUMO system)–deficient mice, significantly delayed retinal vascularization by maintaining prolonged NOTCH1 signaling, as confirmed in cultured endothelial cells. An in vitro SUMOylation assay and immunoprecipitation revealed that when SENP1 associated with N1ICD (NOTCH1 intracellular domain), it functions as a deSUMOylase of N1ICD SUMOylation on conserved lysines. Immunoblot and immunoprecipitation analyses and dual-luciferase assays of natural and SUMO-conjugated/nonconjugated NOTCH1 forms demonstrated that SUMO conjugation facilitated NOTCH1 cleavage. This released N1ICD from the membrane and stabilized it for translocation to the nucleus where it functions as a cotranscriptional factor. Functionally, SENP1-mediated NOTCH1 deSUMOylation was required for NOTCH signal activation in response to DLL4 (Delta-like 4) stimulation. This in turn suppressed VEGF (vascular endothelial growth factor) receptor signaling and angiogenesis, as evidenced by immunoblotted signaling molecules and in vitro angiogenesis assays. Conclusions: These results establish reversible NOTCH1 SUMOylation as a regulatory mechanism in coordinating endothelial angiogenic signaling; SENP1 acts as a critical intrinsic mediator of this process. These findings may apply to NOTCH-regulated biological events in nonvascular tissues and provide a novel therapeutic strategy for vascular diseases and tumors.

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Tobacco Mosaic Virus Movement Protein Functions as a Structural Microtubule-Associated Protein

Journal of Virology ◽

10.1128/jvi.00540-06 ◽

2006 ◽

Vol 80 (17) ◽

pp. 8329-8344 ◽

Cited By ~ 67

Author(s):

Jamie Ashby ◽

Emmanuel Boutant ◽

Mark Seemanpillai ◽

Adrian Sambade ◽

Christophe Ritzenthaler ◽

...

Keyword(s):

Tobacco Mosaic Virus ◽

Mosaic Virus ◽

Movement Protein ◽

Cell Extract ◽

Transport Processes ◽

In Vitro Assays ◽

Protein Functions ◽

Intercellular Spread

ABSTRACT The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

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