RESUMEN
Mammalian glycosaminoglycans (GAGs), except hyaluronan (HA), are sulfated polysaccharides that are covalently attached to core proteins to form proteoglycans (PGs). This article summarizes key biological findings for the most widespread GAGs, namely HA, chondroitin sulfate/dermatan sulfate (CS/DS), keratan sulfate (KS), and heparan sulfate (HS). It focuses on the major processes that remain to be deciphered to get a comprehensive view of the mechanisms mediating GAG biological functions. They include the regulation of GAG biosynthesis and postsynthetic modifications in heparin (HP) and HS, the composition, heterogeneity, and function of the tetrasaccharide linkage region and its role in disease, the functional characterization of the new PGs recently identified by glycoproteomics, the selectivity of interactions mediated by GAG chains, the display of GAG chains and PGs at the cell surface and their impact on the availability and activity of soluble ligands, and on their move through the glycocalyx layer to reach their receptors, the human GAG profile in health and disease, the roles of GAGs and particular PGs (syndecans, decorin, and biglycan) involved in cancer, inflammation, and fibrosis, the possible use of GAGs and PGs as disease biomarkers, and the design of inhibitors targeting GAG biosynthetic enzymes and GAG-protein interactions to develop novel therapeutic approaches.
Asunto(s)
Glicosaminoglicanos , Humanos , Glicosaminoglicanos/metabolismo , Glicosaminoglicanos/química , Animales , Heparitina Sulfato/metabolismo , Heparitina Sulfato/química , Proteoglicanos/metabolismo , Dermatán Sulfato/metabolismo , Dermatán Sulfato/química , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Ácido Hialurónico/metabolismo , Ácido Hialurónico/química , Sulfato de Queratano/metabolismo , Sulfato de Queratano/química , Sulfatos de Condroitina/metabolismo , Sulfatos de Condroitina/químicaRESUMEN
Biglycan, a small leucine-rich proteoglycan (SLRP), is a crucial component of the extracellular matrix (ECM) associated with the maintenance of tissue homeostasis. In response to tissue damage, the ECM-derived soluble form of biglycan acts as a danger signal by triggering an inflammatory response via the toll-like receptor (TLR)2/TLR4 in macrophages and dendritic cells. The impact and signaling mechanism of biglycan in innate immunity is better understood with the use of specific and reliable research tools and investigation techniques. Accordingly, our lab has established explicit and detailed experimental protocols to examine the in vitro and in vivo effects of biglycan in cellular immune responses. To evaluate the in vitro effects of biglycan on macrophage activation, a comprehensive protocol that makes use of murine peritoneal macrophages has been described. Further, to study the in vivo effects of biglycan, a method that uses a pLIVE vector to generate transgenic mice transiently overexpressing human biglycan is detailed. A step-by-step protocol for analyzing the effects of soluble biglycan overexpression in transgenic mice is explained under the following sections: (1) construction of pLIVE-hBGN plasmid, (2) intravenous delivery of transgenic vector, (3) identification of hBGN transgene in hepatocytes (4) detection of transgenic biglycan protein in the plasma of transgenic mice, and (5) evaluation of the presence and pro-inflammatory effects of transgenic biglycan in extrahepatic mouse tissues.
Asunto(s)
Inmunidad Innata , Transducción de Señal , Ratones , Humanos , Animales , Biglicano/genética , Macrófagos/metabolismo , Ratones Transgénicos , Proteínas de la Matriz Extracelular/metabolismoRESUMEN
A20 binding inhibitor of nuclear factor kappa B (NF-κB)-1 (ABIN-1), a polyubiquitin-binding protein, is a signal-induced autophagy receptor that attenuates NF-κB-mediated inflammation and cell death. The present study aimed to elucidate the potential role of ABIN-1 in mitophagy, a biological process whose outcome is decisive in diverse physiological and pathological settings. Microtubule-associated proteins 1A/1B light chain 3B-II (LC3B-II) was found to be in complex with ectopically expressed hemagglutinin (HA)-tagged-full length (FL)-ABIN-1. Bacterial expression of ABIN-1 and LC3A and LC3B showed direct binding of ABIN-1 to LC3 proteins, whereas mutations in the LC3-interacting region (LIR) 1 and 2 motifs of ABIN-1 abrogated ABIN-1/LC3B-II complex formation. Importantly, induction of autophagy in HeLa cells resulted in colocalization of ABIN-1 with LC3B-II in autophagosomes and with lysosomal-associated membrane protein 1 (LAMP-1) in autophagolysosomes, leading to degradation of ABIN-1 with p62. Interestingly, ABIN-1 was found to translocate to damaged mitochondria in HeLa-mCherry-Parkin transfected cells. In line with this observation, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated deletion of ABIN-1 significantly inhibited the degradation of the mitochondrial outer membrane proteins voltage-dependent anion-selective channel 1 (VDAC-1), mitofusin-2 (MFN2), and translocase of outer mitochondrial membrane (TOM)20. In addition, short interfering RNA (siRNA)-mediated knockdown of ABIN-1 significantly decreased lysosomal uptake of mitochondria in HeLa cells expressing mCherry-Parkin and the fluorescence reporter mt-mKEIMA. Collectively, our results identify ABIN-1 as a novel and selective mitochondrial autophagy regulator that promotes mitophagy, thereby adding a new player to the complex cellular machinery regulating mitochondrial homeostasis.
Asunto(s)
Mitocondrias , FN-kappa B , Humanos , FN-kappa B/metabolismo , Células HeLa , Unión Proteica , Mitocondrias/metabolismo , Autofagia , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The mechanisms linking immune responses and inflammation with tumor development are not well understood. Here, we show that the soluble form of the extracellular matrix proteoglycan decorin controls inflammation and tumor growth through PDCD4 (programmed cell death 4) and miR-21 (microRNA-21) by two mechanisms. First, decorin acted as an endogenous ligand of Toll-like receptors 2 and 4 and stimulated production of proinflammatory molecules, including PDCD4, in macrophages. Second, decorin prevented translational repression of PDCD4 by decreasing the activity of transforming growth factor-ß1 and the abundance of oncogenic miR-21, a translational inhibitor of PDCD4. Moreover, increased PDCD4 abundance led to decreased release of the anti-inflammatory cytokine interleukin-10, thereby making the cytokine profile more proinflammatory. This pathway operates in both pathogen-mediated and sterile inflammation, as shown here for sepsis and growth retardation of established tumor xenografts, respectively. Decorin was an early response gene evoked by septic inflammation, and protein concentrations of decorin were increased in the plasma of septic patients and mice. In cancer, decorin reduced the abundance of anti-inflammatory molecules and increased that of proinflammatory molecules, thereby shifting the immune response to a proinflammatory state associated with reduced tumor growth. Thus, by stimulating proinflammatory PDCD4 and decreasing the abundance of miR-21, decorin signaling boosts inflammatory activity in sepsis and suppresses tumor growth.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/metabolismo , Decorina/metabolismo , Macrófagos Peritoneales/metabolismo , MicroARNs/metabolismo , Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Sepsis/metabolismo , Transducción de Señal , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/inmunología , Línea Celular Tumoral , Decorina/genética , Decorina/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-10/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos C3H , Ratones Noqueados , Ratones Desnudos , MicroARNs/genética , MicroARNs/inmunología , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/inmunología , Sepsis/genética , Sepsis/inmunología , Sepsis/patología , Receptor Toll-Like 2/agonistas , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 4/metabolismo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/inmunología , Factor de Crecimiento Transformador beta1/metabolismo , Trasplante HeterólogoRESUMEN
The small leucine-rich proteoglycans (SLRPs) are biologically active components of the extracellular matrix (ECM), consisting of a protein core with leucine rich-repeat (LRR) motifs covalently linked to glycosaminoglycan (GAG) side chains. The diversity in composition resulting from the various combinations of protein cores substituted with one or more GAG chains along with their pericellular localization enables SLRPs to interact with a host of different cell surface receptors, cytokines, growth factors, and other ECM components, leading to modulation of cellular functions. SLRPs are capable of binding to: (i) different types of collagens, thereby regulating fibril assembly, organization, and degradation; (ii) Toll-like receptors (TLRs), complement C1q, and tumor necrosis factor-alpha (TNFalpha), regulating innate immunity and inflammation; (iii) epidermal growth factor receptor (EGF-R), insulin-like growth factor receptor (IGF-IR), and c-Met, influencing cellular proliferation, survival, adhesion, migration, tumor growth and metastasis as well as synthesis of other ECM components; (iv) low-density lipoprotein receptor-related protein (LRP-1) and TGF-beta, modulating cytokine activity and fibrogenesis; and (v) growth factors such as bone morphogenic protein (BMP-4) and Wnt-I-induced secreted protein-1 (WISP-1), controlling cell proliferation and differentiation. Thus, the ability of SLRPs, as ECM components, to directly or indirectly regulate cell-matrix crosstalk, resulting in the modulation of various biological processes, aptly qualifies these compounds as matricellular proteins.