RESUMEN
Transglutaminase 2 (TG2) plays a pivotal role in the pathogenesis of celiac disease (CeD) by deamidating dietary gluten peptides, which facilitates antigenic presentation and a strong anti-gluten T cell response. Here, we elucidate the molecular mechanisms underlying the efficacy of the TG2 inhibitor ZED1227 by performing transcriptional analysis of duodenal biopsies from individuals with CeD on a long-term gluten-free diet before and after a 6-week gluten challenge combined with 100 mg per day ZED1227 or placebo. At the transcriptome level, orally administered ZED1227 effectively prevented gluten-induced intestinal damage and inflammation, providing molecular-level evidence that TG2 inhibition is an effective strategy for treating CeD. ZED1227 treatment preserved transcriptome signatures associated with mucosal morphology, inflammation, cell differentiation and nutrient absorption to the level of the gluten-free diet group. Nearly half of the gluten-induced gene expression changes in CeD were associated with the epithelial interferon-γ response. Moreover, data suggest that deamidated gluten-induced adaptive immunity is a sufficient step to set the stage for CeD pathogenesis. Our results, with the limited sample size, also suggest that individuals with CeD might benefit from an HLA-DQ2/HLA-DQ8 stratification based on gene doses to maximally eliminate the interferon-γ-induced mucosal damage triggered by gluten.
Asunto(s)
Enfermedad Celíaca , Dieta Sin Gluten , Proteínas de Unión al GTP , Perfilación de la Expresión Génica , Glútenes , Mucosa Intestinal , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Enfermedad Celíaca/inmunología , Humanos , Glútenes/inmunología , Transglutaminasas/metabolismo , Transglutaminasas/antagonistas & inhibidores , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/antagonistas & inhibidores , Proteínas de Unión al GTP/genética , Mucosa Intestinal/patología , Mucosa Intestinal/metabolismo , Mucosa Intestinal/inmunología , Mucosa Intestinal/efectos de los fármacos , Femenino , Masculino , Adulto , Transcriptoma , Duodeno/patología , Duodeno/inmunología , Duodeno/metabolismo , Interferón gamma/metabolismo , Persona de Mediana Edad , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Adulto Joven , Inmunidad Adaptativa/efectos de los fármacosAsunto(s)
Enfermedad Celíaca , Proteínas de Unión al GTP , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Humanos , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/inmunología , Transglutaminasas/genética , Transglutaminasas/metabolismo , Transcriptoma , Perfilación de la Expresión Génica , Inhibidores Enzimáticos/uso terapéutico , Inhibidores Enzimáticos/farmacologíaRESUMEN
Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.
Asunto(s)
Calcio , Proteínas de Unión al GTP , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Transglutaminasas/metabolismo , Transglutaminasas/química , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Humanos , Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/química , Cristalografía por Rayos X , Glútenes/metabolismo , Glútenes/química , Modelos Moleculares , Conformación Proteica , Enfermedad Celíaca/metabolismo , Unión ProteicaRESUMEN
Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.
Asunto(s)
Evolución Molecular , Filogenia , Transglutaminasas , Vertebrados , Animales , Transglutaminasas/genética , Transglutaminasas/metabolismo , Vertebrados/genética , Evolución Biológica , Piel/metabolismoRESUMEN
Chemical modifications of histones can mediate diverse DNA-templated processes, including gene transcription1-3. Here we provide evidence for a class of histone post-translational modification, serotonylation of glutamine, which occurs at position 5 (Q5ser) on histone H3 in organisms that produce serotonin (also known as 5-hydroxytryptamine (5-HT)). We demonstrate that tissue transglutaminase 2 can serotonylate histone H3 tri-methylated lysine 4 (H3K4me3)-marked nucleosomes, resulting in the presence of combinatorial H3K4me3Q5ser in vivo. H3K4me3Q5ser displays a ubiquitous pattern of tissue expression in mammals, with enrichment observed in brain and gut, two organ systems responsible for the bulk of 5-HT production. Genome-wide analyses of human serotonergic neurons, developing mouse brain and cultured serotonergic cells indicate that H3K4me3Q5ser nucleosomes are enriched in euchromatin, are sensitive to cellular differentiation and correlate with permissive gene expression, phenomena that are linked to the potentiation of TFIID4-6 interactions with H3K4me3. Cells that ectopically express a H3 mutant that cannot be serotonylated display significantly altered expression of H3K4me3Q5ser-target loci, which leads to deficits in differentiation. Taken together, these data identify a direct role for 5-HT, independent from its contributions to neurotransmission and cellular signalling, in the mediation of permissive gene expression.
Asunto(s)
Regulación de la Expresión Génica , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , Procesamiento Proteico-Postraduccional , Serotonina/metabolismo , Factor de Transcripción TFIID/metabolismo , Animales , Diferenciación Celular , Línea Celular , Femenino , Proteínas de Unión al GTP/metabolismo , Glutamina/química , Glutamina/metabolismo , Humanos , Metilación , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Proteína Glutamina Gamma Glutamiltransferasa 2 , Neuronas Serotoninérgicas/citología , Transglutaminasas/metabolismoRESUMEN
Histone dopaminylation is a newly identified epigenetic mark that plays a role in the regulation of gene transcription, where an isopeptide bond is formed between the fifth amino acid of H3 (i.e., glutamine) and dopamine. Recently, we developed a chemical probe to specifically label and enrich histone dopaminylation via bioorthogonal chemistry. Given this powerful tool, we found that histone H3 glutamine 5 dopaminylation (H3Q5dop) was highly enriched in colorectal tumors, which could be attributed to the high expression level of its regulator, transglutaminase 2 (TGM2), in colon cancer cells. Due to the enzyme promiscuity of TGM2, nonhistone proteins have also been identified as dopaminylation targets; however, the dopaminylated proteome in cancer cells still remains elusive. Here, we utilized our chemical probe to enrich dopaminylated proteins from colorectal cancer cells in a bioorthogonal manner and performed the chemical proteomics analysis. Therefore, 425 dopaminylated proteins were identified, many of which are involved in nucleic acid metabolism and transcription pathways. More importantly, a number of dopaminylation sites were identified and attributed to the successful application of our chemical probe. Overall, these findings shed light on the significant association between cellular protein dopaminylation and cancer development, further suggesting that targeting these pathways may become a promising anticancer strategy.
Asunto(s)
Neoplasias Colorrectales , Histonas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Proteómica , Humanos , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/genética , Proteómica/métodos , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Histonas/metabolismo , Transglutaminasas/metabolismo , Transglutaminasas/genética , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Línea Celular Tumoral , Proteoma/análisis , Proteoma/metabolismo , Procesamiento Proteico-Postraduccional , Glutamina/metabolismo , Glutamina/química , Epigénesis GenéticaRESUMEN
Gaucher disease (GD) is a lysosomal storage disorder (LSD) caused by the defective activity of acid ß-glucosidase (GCase) which results from mutations in GBA1. Neurological forms of GD (nGD) can be generated in mice by intra-peritoneal injection of conduritol B-epoxide (CBE) which irreversibly inhibits GCase. Using this approach, a number of pathological pathways have been identified in mouse brain by RNAseq. However, unlike transcriptomics, proteomics gives direct information about protein expression which is more likely to provide insight into which cellular pathways are impacted in disease. We now perform non-targeted, mass spectrometry-based quantitative proteomics on brains from mice injected with 50 mg/kg body weight CBE for 13 days. Of the 5038 detected proteins, 472 were differentially expressed between control and CBE-injected mice of which 104 were selected for further analysis based on higher stringency criteria. We also compared these proteins with differentially expressed genes (DEGs) identified by RNAseq. Some lysosomal proteins were up-regulated as was interferon signaling, whereas levels of ion channel related proteins and some proteins associated with neurotransmitter signaling were reduced, as was cholesterol metabolism. One protein, transglutaminase 1 (TGM1), which is elevated in a number of neurodegenerative diseases, was absent from the control group but was found at high levels in CBE-injected mice, and located in the extracellular matrix (ECM) in layer V of the cortex and intracellularly in Purkinje cells in the cerebellum. Together, the proteomics data confirm previous RNAseq data and add additional mechanistic understanding about cellular pathways that may play a role in nGD pathology.
Asunto(s)
Enfermedad de Gaucher , Animales , Ratones , Enfermedad de Gaucher/metabolismo , Proteómica , Glucosilceramidasa/genética , Encéfalo/metabolismo , Transglutaminasas/genética , Transglutaminasas/metabolismoRESUMEN
Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.
Asunto(s)
Infecciones por Chlamydia/metabolismo , Chlamydia trachomatis/fisiología , Proteínas de Unión al GTP/metabolismo , Regulación Enzimológica de la Expresión Génica , Glucosamina/metabolismo , Glucosa/metabolismo , Hexosaminas/biosíntesis , Transglutaminasas/metabolismo , Animales , Transporte Biológico , Infecciones por Chlamydia/microbiología , Células Epiteliales/metabolismo , Fibroblastos , Fructosafosfatos/metabolismo , Proteínas de Unión al GTP/genética , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas/genéticaRESUMEN
Microbial transglutaminase (MTG) from Streptomyces mobaraensis is a powerful biocatalytic glue for site-specific cross-linking of a range of biomolecules and synthetic molecules that have an MTG-reactive moiety. The preparation of active recombinant MTG requires post-translational proteolytic digestion of a propeptide that functions as an intramolecular chaperone to assist the correct folding of the MTG zymogen (MTGz) in the biosynthesis. Herein, we report engineered active zymogen of MTG (EzMTG) that is expressed in soluble form in the host Escherichia coli cytosol and exhibits cross-linking activity without limited proteolysis of the propeptide. We found that the saturation mutagenesis of residues K10 or Y12 in the propeptide domain generated several active MTGz mutants. In particular, the K10D/Y12G mutant exhibited catalytic activity comparable to that of mature MTG. However, the expression level was low, possibly because of decreased chaperone activity and/or the promiscuous substrate specificity of MTG, which is potentially harmful to the host cells. The K10R/Y12A mutant exhibited specific substrate-dependent reactivity toward peptidyl substrates. Quantitative analysis of the binding affinity of the mutated propeptides to the active site of MTG suggested an inverse relationship between the binding affinity and the catalytic activity of EzMTG. Our proof-of-concept study provides insights into the design of a new biocatalyst using the MTGz as a scaffold and a potential route to high-throughput screening of EzMTG mutants for bioconjugation applications.
Asunto(s)
Precursores Enzimáticos , Transglutaminasas , Precursores Enzimáticos/genética , Transglutaminasas/metabolismoRESUMEN
A versatile chemo-enzymatic tool to site-specifically modify native (nonengineered) antibodies is using transglutaminase (TGase, E.C. 2.3.2.13). With various amines as cosubstrates, this enzyme converts the unsubstituted side chain amide of glutamine (Gln or Q) in peptides and proteins into substituted amides (i.e., conjugates). A pleasant surprise is that only a single conserved glutamine (Gln295) in the Fc region of IgG is modified by microbial TGase (mTGase, EC 2.3.2.13), thereby providing a highly specific and generally applicable conjugation method. However, prior to the transamidation (access to the glutamine residue by mTGase), the steric hindrance from the nearby conserved N-glycan (Asn297 in IgG1) must be reduced. In previous approaches, amidase (PNGase F, EC 3.5.1.52) was used to completely remove the N-glycan. However, PNGase F also converts a net neutral asparagine (Asn297) to a negatively charged aspartic acid (Asp297). This charge alteration may markedly change the structure, function, and immunogenicity of an IgG antibody. In contrast, in our new method presented herein, the N-glycan is trimmed by an endoglycosidase (EndoS2, EC 3.2.1.96), hence retaining both the core N-acetylglucosamine (GlcNAc) moiety and the neutral asparaginyl amide. The trimmed glycan also reduces or abolishes Fc receptor-mediated functions, which results in better imaging agents by decreasing nonspecific binding to other cells (e.g., immune cells). Moreover, the remaining core glycan allows further derivatization such as glycan remodeling and dual conjugation. Practical and robust, our method generates conjugates in near quantitative yields, and both enzymes are commercially available.
Asunto(s)
Glutamina , Glicósido Hidrolasas , Glutamina/química , Péptido-N4-(N-acetil-beta-glucosaminil) Asparagina Amidasa , Transglutaminasas/metabolismo , Inmunoglobulina G/química , Polisacáridos/química , AmidasRESUMEN
The preservation of the native conformation and functionality of membrane proteins has posed considerable challenges. While detergents and liposome reconstitution have been traditional approaches, nanodiscs (NDs) offer a promising solution by embedding membrane proteins in phospholipids encircled by an amphipathic helical protein MSP belt. Nevertheless, a drawback of commonly used NDs is their limited homogeneity and stability. In this study, we present a novel approach to construct covalent annular nanodiscs (cNDs) by leveraging microbial transglutaminase (MTGase) to catalyze isopeptide bond formation between the side chains of terminal amino acids, specifically Lysine (K) and Glutamine (Q). This methodology significantly enhances the homogeneity and stability of NDs. Characterization of cNDs and the assembly of membrane proteins within them validate the successful reconstitution of membrane proteins with improved homogeneity and stability. Our findings suggest that cNDs represent a more suitable tool for investigating interactions between membrane proteins and lipids, as well as for analyzing membrane protein structures.
Asunto(s)
Proteínas de la Membrana , Nanoestructuras , Transglutaminasas , Nanoestructuras/química , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Transglutaminasas/química , Transglutaminasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismoRESUMEN
The decellularized tilapia skin (dTS) has gained significant attention as a promising material for tissue regeneration due to its ability to provide unique structural and functional components that support cell growth, adhesion, and proliferation. However, the clinical application of dTS is limited by its low mechanical strength and rapid biodegradability. Herein, we prepare a novel RGD (arginine-glycine-aspartic acid) functionalized dTS scaffold (dTS/RGD) by using transglutaminase (TGase) crosslinking. The developed dTS/RGD scaffold possesses excellent properties, including a medium porosity of â¼59.2%, a suitable degradation rate of approximately 80% over a period of two weeks, and appropriate mechanical strength with a maximum tensile stress of â¼46.36 MPa which is much higher than that of dTS (â¼32.23 MPa). These properties make the dTS/RGD scaffold ideal for promoting cell adhesion and proliferation, thereby accelerating skin wound healing in a full-thickness skin defect model. Such an enzymatic cross-linking strategy provides a favorable microenvironment for wound healing and holds great potential for application in skin regeneration engineering.
Asunto(s)
Oligopéptidos , Regeneración , Piel , Tilapia , Andamios del Tejido , Transglutaminasas , Animales , Andamios del Tejido/química , Tilapia/metabolismo , Transglutaminasas/metabolismo , Transglutaminasas/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Cicatrización de Heridas , Proliferación Celular , Ingeniería de Tejidos , Porosidad , Ratones , Adhesión Celular , HumanosRESUMEN
Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.
Asunto(s)
Antineoplásicos , Carcinoma de Células Renales , Neoplasias Renales , Proteína Glutamina Gamma Glutamiltransferasa 2 , Animales , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Imidazoles/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Neoplasias Renales/patología , Simulación del Acoplamiento Molecular , Proteína Glutamina Gamma Glutamiltransferasa 2/antagonistas & inhibidores , Transglutaminasas/antagonistas & inhibidores , Transglutaminasas/metabolismo , Proteína p53 Supresora de Tumor/efectos de los fármacos , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Porcine placental extract (PPE) is commonly used in various health foods and cosmetics. PPE use in cosmetics predominantly consist of the water-soluble fraction derived from the entire placenta. In this report, we examined the effect of the hydrophobic constituents of the PPE, specifically the sphingolipid-enriched fraction designated as the sphingolipid-enriched porcine placental extract (SLPPE), on the expression of genes associated with skin function in cultured normal human epidermal keratinocytes. Using quantitative RT-PCR (qRT-PCR) analysis, we found that SLPPE concentrations ranging from 25 to 100 µg/mL upregulated the gene expression of key components associated with the cornified envelope structure (filaggrin (FLG), involucrin (IVL) and loricrin (LOR)), cornification enzymes (transglutaminase 1 (TGM1) and TGM5) and the desquamation enzymes (kallikrein 5 (KLK5) and KLK7). Additionally, KLK5p and FLG protein (FLGp) were detected in the culture supernatants of keratinocytes treated with SLPPE at these concentrations. These findings suggest that SLPPE is possible to promote the cornification and desquamation in epidermal keratinocytes, and it may offer potential benefits in cosmetics.
Asunto(s)
Proteínas Filagrina , Calicreínas , Queratinocitos , Esfingolípidos , Transglutaminasas , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Humanos , Animales , Transglutaminasas/metabolismo , Transglutaminasas/genética , Porcinos , Esfingolípidos/metabolismo , Calicreínas/metabolismo , Calicreínas/genética , Extractos Placentarios/farmacología , Células Cultivadas , Femenino , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , EmbarazoRESUMEN
One of the major mysteries in science is how it is possible to pack the cellular chromatin with a total length of over 1 m, into a small sphere with a diameter of 5 mm "the nucleus", and even more difficult to envisage how to make it functional. Although we know that compaction is achieved through the histones, however, the DNA needs to be accessible to the transcription machinery and this is allowed thanks to a variety of very complex epigenetic mechanisms. Either DNA (methylation) or post-translational modifications of histone proteins (acetylation, methylation, ubiquitination and sumoylation) play a crucial role in chromatin remodelling and consequently on gene expression. Recently the serotonylation and dopaminylation of the histone 3, catalyzed by the Transglutaminase type 2 (TG2), has been reported. These novel post-translational modifications catalyzed by a predominantly cytoplasmic enzyme opens a new avenue for future investigations on the enzyme function itself and for the possibility that other biological amines, substrate of TG2, can influence the genome regulation under peculiar cellular conditions. In this review we analyzed the nuclear TG2's biology by discussing both its post-translational modification of various transcription factors and the implications of its epigenetic new face. Finally, we will focus on the potential impact of these events in human diseases.
Asunto(s)
Ensamble y Desensamble de Cromatina , Citoplasma , Epigénesis Genética , Histonas , Transglutaminasas , Humanos , Acetilación , Cromatina , ADN/genética , Metilación de ADN , Histonas/metabolismo , Procesamiento Proteico-Postraduccional , Transglutaminasas/genética , Transglutaminasas/metabolismo , Citoplasma/enzimología , Citoplasma/genética , Citoplasma/metabolismo , Núcleo Celular/enzimología , Núcleo Celular/genética , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/fisiologíaRESUMEN
Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.
Asunto(s)
ADN Complementario , Secuenciación de Nucleótidos de Alto Rendimiento , Transglutaminasas , Humanos , Transglutaminasas/genética , Transglutaminasas/metabolismo , Especificidad por Sustrato , ADN Complementario/genética , Secuencia de Aminoácidos , Biblioteca de PéptidosRESUMEN
Transglutaminase type 2 (TG2) is the most ubiquitously expressed and well characterized member of the transglutaminase family. It is a ubiquitous multifunctional enzyme implicated in the regulation of several cellular pathways that support the survival, death, and general homeostasis of eukaryotic cells. Due to its multiple localizations both inside and outside the cell, TG2 participates in the regulation of many crucial intracellular signaling cascades in a tissue- and cell-specific manner, making this enzyme an important player in disease development and progression. Moreover, TG2 is capable of modulating the tumor microenvironment, a process of dynamic tissue remodeling and biomechanical events, resulting in changes which influence tumor initiation, growth, and metastasis. Even if generally related to the Ca2+-dependent post-translational modification of proteins, a number of different biological functions have been ascribed to TG2, like those of a peptide isomerase, protein kinase, guanine nucleotide binder, and cytosolic-nuclear translocator. With respect to cancer, TG2's role is controversial and highly debated; it has been described both as an anti- and pro-apoptotic factor and is linked to all the processes of tumorigenesis. However, numerous pieces of evidence support a tissue-specific role of TG2 so that it can assume both oncogenic and tumor-suppressive roles.
Asunto(s)
Neoplasias , Proteína Glutamina Gamma Glutamiltransferasa 2 , Humanos , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Transducción de Señal , Microambiente TumoralRESUMEN
Neurodegenerative diseases encompass a heterogeneous group of disorders that afflict millions of people worldwide. Characteristic protein aggregates are histopathological hallmark features of these disorders, including Amyloid ß (Aß)-containing plaques and tau-containing neurofibrillary tangles in Alzheimer's disease, α-Synuclein (α-Syn)-containing Lewy bodies and Lewy neurites in Parkinson's disease and dementia with Lewy bodies, and mutant huntingtin (mHTT) in nuclear inclusions in Huntington's disease. These various aggregates are found in specific brain regions that are impacted by neurodegeneration and associated with clinical manifestations. Transglutaminase (TG2) (also known as tissue transglutaminase) is the most ubiquitously expressed member of the transglutaminase family with protein crosslinking activity. To date, Aß, tau, α-Syn, and mHTT have been determined to be substrates of TG2, leading to their aggregation and implicating the involvement of TG2 in several pathophysiological events in neurodegenerative disorders. In this review, we summarize the biochemistry and physiologic functions of TG2 and describe recent advances in the pathogenetic role of TG2 in these diseases. We also review TG2 inhibitors tested in clinical trials and discuss recent TG2-targeting approaches, which offer new perspectives for the design of future highly potent and selective drugs with improved brain delivery as a disease-modifying treatment for neurodegenerative disorders.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Enfermedades Neurodegenerativas/metabolismo , Proteína Glutamina Gamma Glutamiltransferasa 2 , Péptidos beta-Amiloides , alfa-Sinucleína/metabolismo , Enfermedad de Parkinson/metabolismo , Transglutaminasas/metabolismo , Proteínas tauRESUMEN
Glioblastoma (GBM) is one of the most aggressive cancers, characterized by a decrease in antioxidant levels. Evidence has demonstrated that ferulic acid (FA), a natural antioxidant particularly abundant in vegetables and fruits, could be a promising candidate for GBM treatment. Since FA shows a high instability that compromises its therapeutic application, it has been encapsulated into Nanostructured Lipid Carriers (NLCs) to improve its bioavailability in the brain. It has been demonstrated that tissue transglutaminase (TG2) is a multi-functional protein implicated in many physiological and pathological processes, including cancer. TG2 is also involved in GBM correlated with metastasis formation and drug resistance. Therefore, the evaluation of TG2 expression levels and its cellular localization are important to assess the anti-cancer effect of FA against GBM cancer. Our results have demonstrated that treatment with free FA and FA-NLCs in the U87-MG cancer cell line differently modified TG2 localization and expression levels. In the cells treated with free FA, TG2 appeared expressed both in the cytosol and in the nucleus, while the treatment with FA-NLCs showed that the protein is exclusively localized in the cytosol, exerting its pro-apoptotic effect. Therefore, our data suggest that FA loaded in NLCs could represent a promising natural agent for supplementing the current anti-cancer drugs used for the treatment of GBM.
Asunto(s)
Ácidos Cumáricos , Proteínas de Unión al GTP , Glioblastoma , Nanopartículas , Proteína Glutamina Gamma Glutamiltransferasa 2 , Transglutaminasas , Ácidos Cumáricos/farmacología , Humanos , Transglutaminasas/metabolismo , Transglutaminasas/genética , Glioblastoma/metabolismo , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Proteína Glutamina Gamma Glutamiltransferasa 2/metabolismo , Línea Celular Tumoral , Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/genética , Nanopartículas/química , Portadores de Fármacos/química , Apoptosis/efectos de los fármacos , Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacosRESUMEN
Transglutaminase (TGase)-catalyzed crosslinking has gained substantial traction as a novel strategy for reducing allergenic risk in food proteins, particularly within the realm of hypoallergenic food production. This study explored the impact of TGase crosslinking on conformational changes in a binary protein system composed of soy protein isolate (SPI) and sodium caseinate (SC) at varying mass ratios (10:0, 7:3, 5:5, 3:7 (w/w)). Specifically, the immunoglobulin E (IgE) binding capacity of soy proteins within this system was examined. Prolonged TGase crosslinking (ranging from 0 h to 15 h) resulted in a gradual reduction in IgE reactivity across all SPI-SC ratios, with the order of IgE-binding capability as follows: SPI > SPI5-SC5 > SPI7-SC3 > SPI3-SC7. These alterations in protein conformation following TGase crosslinking, as demonstrated by variable intrinsic fluorescence, altered surface hydrophobicity, increased ultraviolet absorption and reduced free sulfhydryl content, were identified as the underlying causes. Additionally, ionic bonds were found to play a significant role in maintaining the structure of the dual-protein system after crosslinking, with hydrophobic forces and hydrogen bonds serving as supplementary forces. Generally, the dual-protein system may exhibit enhanced efficacy in reducing the allergenicity of soy protein.