RESUMEN
Transmembrane prolyl 4-hydroxylase (P4H-TM) is an enigmatic enzyme whose cellular function and primary substrate remain to be identified. Its loss-of-function mutations cause a severe neurological HIDEA syndrome with hypotonia, intellectual disability, dysautonomia and hypoventilation. Previously, P4H-TM deficiency in mice was associated with reduced atherogenesis and lower serum triglyceride levels. Here, we characterized the glucose and lipid metabolism of P4h-tm-/- mice in physiological and tissue analyses. P4h-tm-/- mice showed variations in 24-h oscillations of energy expenditure, VO2 and VCO2 and locomotor activity compared to wild-type (WT) mice. Their rearing activity was reduced, and they showed significant muscle weakness and compromised coordination. Sedated P4h-tm-/- mice had better glucose tolerance, lower fasting insulin levels, higher fasting lactate levels and lower fasting free fatty acid levels compared to WT. These alterations were not present in conscious P4h-tm-/- mice. Fasted P4h-tm-/- mice presented with faster hepatic glycogenolysis. The respiratory rate of conscious P4h-tm-/- mice was significantly lower compared to the WT, the decrease being further exacerbated by sedation and associated with acidosis and a reduced ventilatory response to both hypoxia and hypercapnia. P4H-TM deficiency in mice is associated with alterations in whole-body energy metabolism, day-night rhythm of activity, glucose homeostasis and neuromuscular and respiratory functions. Although the underlying mechanism(s) are not yet fully understood, the phenotype appears to have neurological origins, controlled by brain and central nervous system circuits. The phenotype of P4h-tm-/- mice recapitulates some of the symptoms of HIDEA patients, making this mouse model a valuable tool to study and develop tailored therapies.
Asunto(s)
Metabolismo Energético , Animales , Ratones , Ratones Endogámicos C57BL , Masculino , Metabolismo de los Lípidos , Prolil Hidroxilasas/metabolismo , Glucosa/metabolismo , Ratones Noqueados , Hígado/metabolismoRESUMEN
Collagen biosynthesis requires several co- and post-translational modifications of lysine and proline residues to form structurally and functionally competent collagen molecules. Formation of 4-hydroxyproline (4Hyp) in Y-position prolines of the repetitive -X-Y-Gly- sequences provides thermal stability for the triple-helical collagen molecules. 4Hyp formation is catalyzed by a collagen prolyl 4-hydroxylase (C-P4H) family consisting of three isoenzymes. Here we identify specific roles for the two main C-P4H isoenzymes in collagen hydroxylation by a detailed 4Hyp analysis of type I and IV collagens derived from cell and tissue samples. Loss of C-P4H-I results in underhydroxylation of collagen where the affected prolines are not uniformly distributed, but mainly present in sites where the adjacent X-position amino acid has a positively charged or a polar uncharged side chain. In contrast, loss of C-P4H-II results in underhydroxylation of triplets where the X-position is occupied by a negatively charged amino acid glutamate or aspartate. Hydroxylation of these triplets was found to be important as loss of C-P4H-II alone resulted in reduced collagen melting temperature and altered assembly of collagen fibrils and basement membrane. The observed C-P4H isoenzyme differences in substrate specificity were explained by selective binding of the substrate to the active site resulting in distinct differences in Km and Vmax values. Furthermore, our results clearly show that the substrate proline selection is not dependent on the collagen type, but the main determinant is the X-position amino acid of the -X-Pro-Gly- triplet. Although our data clearly shows the necessity of both C-P4H-I and II for normal prolyl 4-hydroxylation and function of collagens, the mRNA expression of the isoenzymes with various procollagens was, surprisingly, not tightly coordinated, suggesting additional levels of control. In conclusion, this study provides a molecular level explanation for the need of multiple C-P4H isoenzymes to generate collagen molecules capable to assemble into intact extracellular matrix structures.
Asunto(s)
Dipéptidos , Isoenzimas , Prolil Hidroxilasas , Prolil Hidroxilasas/genética , Isoenzimas/genética , Colágeno Tipo I/genética , Procolágeno-Prolina Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/química , Procolágeno-Prolina Dioxigenasa/metabolismo , Colágeno/genética , Colágeno/metabolismo , Prolina/metabolismoRESUMEN
BACKGROUND: Collagen XIII is a transmembrane collagen associated with neuromuscular junction development, and in humans its deficiency results in congenital myasthenic syndrome type 19 (CMS19), which leads to breathing difficulties. CMS19 patients usually have restricted lung capacity and one patient developed chronic lung disease. In single-cell RNA sequencing studies, collagen XIII has been identified as a marker for pulmonary lipofibroblasts, which have been implicated in the resolution of pulmonary fibrosis. METHODS: We investigated the location and function of collagen XIII in the lung to understand the origin of pulmonary symptoms in human CMS19 patients. Additionally, we performed immunostainings on idiopathic pulmonary fibrosis (IPF) samples (N=5) and both normal and fibrotic mouse lung. To study whether the lack of collagen XIII predisposes to restrictive lung disease, we exposed Col13a1-modified mice to bleomycin-induced pulmonary fibrosis. RESULTS: Apparently normal alveolar septum sections of IPF patients' lungs stained faintly for collagen XIII, and its expression was pinpointed to the septal fibroblasts in the mouse lung. Lung capacity was increased in mice lacking collagen XIII by over 10%. In IPF samples, collagen XIII was expressed by basal epithelial cells, hyperplastic alveolar epithelial cells and stromal cells in fibrotic areas, but the development of pulmonary fibrosis was unaffected in collagen XIII-deficient mice. CONCLUSIONS: Changes in mouse lung function appear to represent a myasthenic manifestation of collagen XIII deficiency. We suggest that respiratory muscle myasthenia is the primary cause of the breathing problems suffered by CMS19 patients in addition to skeletal deformities. Induction of collagen XIII expression in the IPF patients' lungs warrants further studies to reveal collagen XIII-dependent disease mechanisms.
Asunto(s)
Fibrosis Pulmonar Idiopática , Fenómenos Fisiológicos Respiratorios , Humanos , Animales , Ratones , Disnea , Colágeno , PulmónRESUMEN
Collagen prolyl 4-hydroxylases (C-P4H) are α2ß2 tetramers, which catalyze the prolyl 4-hydroxylation of procollagen, allowing for the formation of the stable triple-helical collagen structure in the endoplasmic reticulum. The C-P4H α-subunit provides the N-terminal dimerization domain, the middle peptide-substrate-binding (PSB) domain, and the C-terminal catalytic (CAT) domain, whereas the ß-subunit is identical to the enzyme protein disulfide isomerase (PDI). The structure of the N-terminal part of the α-subunit (N-terminal region and PSB domain) is known, but the structures of the PSB-CAT linker region and the CAT domain as well as its mode of assembly with the ß/PDI subunit, are unknown. Here, we report the crystal structure of the CAT domain of human C-P4H-II complexed with the intact ß/PDI subunit, at 3.8 Å resolution. The CAT domain interacts with the a, b', and a' domains of the ß/PDI subunit, such that the CAT active site is facing bulk solvent. The structure also shows that the C-P4H-II CAT domain has a unique N-terminal extension, consisting of α-helices and a ß-strand, which is the edge strand of its major antiparallel ß-sheet. This extra region of the CAT domain interacts tightly with the ß/PDI subunit, showing that the CAT-PDI interface includes an intersubunit disulfide bridge with the a' domain and tight hydrophobic interactions with the b' domain. Using this new information, the structure of the mature C-P4H-II α2ß2 tetramer is predicted. The model suggests that the CAT active-site properties are modulated by α-helices of the N-terminal dimerization domains of both subunits of the α2-dimer.
Asunto(s)
Prolil Hidroxilasas , Proteína Disulfuro Isomerasas , Humanos , Dominio Catalítico , Colágeno/metabolismo , Péptidos/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolil Hidroxilasas/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Conformación ProteicaRESUMEN
Alzheimer's disease (AD) is the most common cause of dementia with limited treatment options affecting millions of people and the prevalence increasing with the aging population. The current knowledge on the role of the hypoxia/hypoxia-inducible factor (HIF) in the AD pathology is restricted and controversial. We hypothesized based on benefits of the genetic long-term inactivation of HIF prolyl 4-hydroxylase-2 (HIF-P4H-2) on metabolism, vasculature and inflammatory response that prolonged moderate activation of the hypoxia response could hinder AD pathology. We used an aging model to study potential spontaneous accumulation of amyloid-ß (Aß) in HIF-P4H-2-deficient mice and a transgenic APP/PSEN1 mouse model subjected to prolonged sustained environmental hypoxia (15% O2 for 6 weeks) at two different time points of the disease; at age of 4 and 10 months. In both settings, activation of the hypoxia response reduced brain protein aggregate levels and this associated with higher vascularity. In the senescent HIF-P4H-2-deficient mice metabolic reprogramming also contributed to less protein aggregates while in APP/PSEN1 mice lesser Aß associated additionally with hypoxia-mediated favorable responses to neuroinflammation and amyloid precursor protein processing. In conclusion, continuous, non-full-scale activation of the HIF pathway appears to mediate protection against neurodegeneration via several mechanisms and should be studied as a treatment option for AD.
Asunto(s)
Enfermedad de Alzheimer , Precursor de Proteína beta-Amiloide , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Hipoxia/genética , Ratones , Ratones TransgénicosRESUMEN
Hypoxia-inducible factor (HIF) prolyl 4-hydroxylases (HIF-P4Hs 1-3) are druggable targets in renal anemia, where pan-HIF-P4H inhibitors induce an erythropoietic response. Preclinical data suggest that HIF-P4Hs could also be therapeutic targets for treating metabolic dysfunction, although the contributions of HIF-P4H isoenzymes in various tissues to the metabolic phenotype are inadequately understood. Here, we used mouse lines that were gene-deficient for HIF-P4Hs 1 to 3 and two preclinical pan-HIF-P4H inhibitors to study the contributions of these isoenzymes to the anthropometric and metabolic outcome and HIF response. We show both inhibitors induced a HIF response in wildtype white adipose tissue (WAT), liver, and skeletal muscle and alleviated metabolic dysfunction during a 6-week treatment period, but they did not alter healthy metabolism. Our data indicate that HIF-P4H-1 contributed especially to skeletal muscle and WAT metabolism and that its loss lowered body weight and serum cholesterol levels upon aging. In addition, we found HIF-P4H-3 had effects on the liver and WAT and its loss increased body weight, adiposity, liver weight and triglyceride levels, WAT inflammation, and cholesterol levels and resulted in hyperglycemia and insulin resistance, especially during aging. Finally, we demonstrate HIF-P4H-2 affected all tissues studied; its inhibition lowered body and liver weight and serum cholesterol levels and improved glucose tolerance. We found very few HIF target metabolic mRNAs were regulated by the inhibition of three isoenzymes, thus suggesting a potential for selective therapeutic tractability. Altogether, these data provide specifications for the future development of HIF-P4H inhibitors for the treatment of metabolic diseases.
Asunto(s)
Prolina Dioxigenasas del Factor Inducible por Hipoxia , Isoenzimas , Tejido Adiposo Blanco/metabolismo , Envejecimiento/metabolismo , Animales , Peso Corporal , Colesterol/sangre , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/antagonistas & inhibidores , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Resistencia a la Insulina , Isoenzimas/antagonistas & inhibidores , Isoenzimas/metabolismo , Hígado/metabolismo , Ratones , Músculo Esquelético/metabolismo , Obesidad/metabolismoRESUMEN
Proper deposition of the extracellular matrix and its major components, the collagens, is essential for endochondral ossification and bone mass accrual. Collagen prolyl 4-hydroxylases (C-P4Hs) hydroxylate proline residues in the -X-Pro-Gly- repeats of all known collagen types. Their product, 4-hydroxyproline, is essential for correct folding and thermal stability of the triple-helical collagen molecules in physiological body temperatures. We have previously shown that inactivation of the mouse P4ha1 gene, which codes for the catalytic α subunit of the major C-P4H isoform, is embryonic lethal, whereas inactivation of the P4ha2 gene produced only a minor phenotype. Instead, mice with a haploinsufficiency of the P4ha1 gene combined with a homozygous deletion of the P4ha2 gene present with a moderate chondrodysplasia due to transient cell death of the growth plate chondrocytes. Here, to further characterize the bone phenotype of the P4ha1 +/-; P4ha2 -/- mice, we have carried out gene expression analyses at whole-tissue and single-cell levels, biochemical analyses, microcomputed tomography, histomorphometric analyses, and second harmonic generation microscopy to show that C-P4H α subunit expression peaks early and that the C-P4H deficiency leads to reduced collagen amount, a reduced rate of bone formation, and a loss of trabecular and cortical bone volume in the long bones. The total osteoblast number in the proximal P4ha1 +/-; P4ha2 -/- tibia and the C-P4H activity in primary P4ha1 +/-; P4ha2 -/- osteoblasts were reduced, whereas the population of osteoprogenitor colony-forming unit fibroblasts was increased in the P4ha1 +/-; P4ha2 -/- marrow. Thus, the P4ha1 +/-; P4ha2 -/- mouse model recapitulates key aspects of a recently recognized congenital connective tissue disorder with short stature and bone dysplasia caused by biallelic variants of the human P4HA1 gene. Altogether, the data demonstrate the allele dose-dependent importance of the C-P4Hs to the developing organism and a threshold effect of C-P4H activity in the proper production of bone matrix. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
RESUMEN
Hypoxia-inducible factors (HIFs) induce numerous genes regulating oxygen homeostasis. As oxygen sensors of the cells, the HIF prolyl 4-hydroxylases (HIF-P4Hs) regulate the stability of HIFs in an oxygen-dependent manner. During hair follicle (HF) morphogenesis and cycling, the location of dermal papilla (DP) alternates between the dermis and hypodermis and results in varying oxygen levels for the DP cells. These cells are known to express hypoxia-inducible genes, but the role of the hypoxia response pathway in HF development and homeostasis has not been studied. Using conditional gene targeting and analysis of hair morphogenesis, we show here that lack of Hif-p4h-2 in Forkhead box D1 (FoxD1)-lineage mesodermal cells interferes with the normal HF development in mice. FoxD1-lineage cells were found to be mainly mesenchymal cells located in the dermis of truncal skin, including those cells composing the DP of HFs. We found that upon Hif-p4h-2 inactivation, HF development was disturbed during the first catagen leading to formation of epithelial-lined HF cysts filled by unorganized keratins, which eventually manifested as truncal alopecia. Furthermore, the depletion of Hif-p4h-2 led to HIF stabilization and dysregulation of multiple genes involved in keratin formation, HF differentiation, and HIF, transforming growth factor ß (TGF-ß), and Notch signaling. We hypothesize that the failure of HF cycling is likely to be mechanistically caused by disruption of the interplay of the HIF, TGF-ß, and Notch pathways. In summary, we show here for the first time that HIF-P4H-2 function in FoxD1-lineage cells is essential for the normal development and homeostasis of HFs.
Asunto(s)
Alopecia , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Alopecia/enzimología , Alopecia/genética , Animales , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , Oxígeno/metabolismo , Factor de Crecimiento Transformador betaRESUMEN
Hypoxia-inducible factor prolyl 4-hydroxylases (HIF-P4Hs) regulate the hypoxic induction of >300 genes required for survival and adaptation under oxygen deprivation. Inhibition of HIF-P4H-2 has been shown to be protective in focal cerebral ischemia rodent models, while that of HIF-P4H-1 has no effects and inactivation of HIF-P4H-3 has adverse effects. A transmembrane prolyl 4-hydroxylase (P4H-TM) is highly expressed in the brain and contributes to the regulation of HIF, but the outcome of its inhibition on stroke is yet unknown. To study this, we subjected WT and P4htm-/- mice to permanent middle cerebral artery occlusion (pMCAO). Lack of P4H-TM had no effect on lesion size following pMCAO, but increased inflammatory microgliosis and neutrophil infiltration was observed in the P4htm-/- cortex. Furthermore, both the permeability of blood brain barrier and ultrastructure of cerebral tight junctions were compromised in P4htm-/- mice. At the molecular level, P4H-TM deficiency led to increased expression of proinflammatory genes and robust activation of protein kinases in the cortex, while expression of tight junction proteins and the neuroprotective growth factors erythropoietin and vascular endothelial growth factor was reduced. Our data provide the first evidence that P4H-TM inactivation has no protective effect on infarct size and increases inflammatory microgliosis and neutrophil infiltration in the cortex at early stage after pMCAO. When considering HIF-P4H inhibitors as potential therapeutics in stroke, the current data support that isoenzyme-selective inhibitors that do not target P4H-TM or HIF-P4H-3 would be preferred.
Asunto(s)
Barrera Hematoencefálica , Infarto de la Arteria Cerebral Media , Enfermedades Neuroinflamatorias , Prolil Hidroxilasas , Accidente Cerebrovascular , Animales , Barrera Hematoencefálica/enzimología , Barrera Hematoencefálica/metabolismo , Permeabilidad de la Membrana Celular , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Infarto de la Arteria Cerebral Media/enzimología , Infarto de la Arteria Cerebral Media/metabolismo , Ratones , Enfermedades Neuroinflamatorias/enzimología , Enfermedades Neuroinflamatorias/metabolismo , Permeabilidad , Prolil Hidroxilasas/metabolismo , Inhibidores de Prolil-Hidroxilasa/farmacología , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Hypoxia plays an important regulatory role in the vasculature to adjust blood flow to meet metabolic requirements. At the level of gene transcription, the responses are mediated by hypoxia-inducible factor (HIF) the stability of which is controlled by the HIF prolyl 4-hydroxylase-2 (PHD2). In the lungs hypoxia results in vasoconstriction, however, the pathophysiological relevance of PHD2 in the major arterial cell types; endothelial cells (ECs) and arterial smooth muscle cells (aSMCs) in the adult vasculature is incompletely characterized. Here, we investigated PHD2-dependent vascular homeostasis utilizing inducible deletions of PHD2 either in ECs (Phd2∆ECi) or in aSMCs (Phd2∆aSMC). Cardiovascular function and lung pathologies were studied using echocardiography, Doppler ultrasonography, intraventricular pressure measurement, histological, ultrastructural, and transcriptional methods. Cell intrinsic responses were investigated in hypoxia and in conditions mimicking hypertension-induced hemodynamic stress. Phd2∆ECi resulted in progressive pulmonary disease characterized by a thickened respiratory basement membrane (BM), alveolar fibrosis, increased pulmonary artery pressure, and adaptive hypertrophy of the right ventricle (RV). A low oxygen environment resulted in alterations in cultured ECs similar to those in Phd2∆ECi mice, involving BM components and vascular tone regulators favoring the contraction of SMCs. In contrast, Phd2∆aSMC resulted in elevated RV pressure without alterations in vascular tone regulators. Mechanistically, PHD2 inhibition in aSMCs involved actin polymerization -related tension development via activated cofilin. The results also indicated that hemodynamic stress, rather than PHD2-dependent hypoxia response alone, potentiates structural remodeling of the extracellular matrix in the pulmonary microvasculature and respiratory failure.
Asunto(s)
Hipertensión Pulmonar , Animales , Arterias/metabolismo , Células Endoteliales/metabolismo , Fibrosis , Hipertensión Pulmonar/genética , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , Miocitos del Músculo Liso/patología , Prolil Hidroxilasas/metabolismoRESUMEN
AIMS: We have previously demonstrated protection against obesity, metabolic dysfunction, atherosclerosis and cardiac ischemia in a hypoxia-inducible factor (HIF) prolyl 4-hydroxylase-2 (Hif-p4h-2) deficient mouse line, attributing these protective effects to activation of the hypoxia response pathway in a normoxic environment. We intended here to find out whether the Hif-p4h-2 deficiency affects the cardiac health of these mice upon aging. METHODS AND RESULTS: When the Hif-p4h-2 deficient mice and their wild-type littermates were monitored during normal aging, the Hif-p4h-2 deficient mice had better preserved diastolic function than the wild type at one year of age and less cardiomyocyte hypertrophy at two years. On the mRNA level, downregulation of hypertrophy-associated genes was detected and shown to be associated with upregulation of Notch signaling, and especially of the Notch target gene and transcriptional repressor Hairy and enhancer-of-split-related basic helix-loop-helix (Hey2). Blocking of Notch signaling in cardiomyocytes isolated from Hif-p4h-2 deficient mice with a gamma-secretase inhibitor led to upregulation of the hypertrophy-associated genes. Also, targeting Hey2 in isolated wild-type rat neonatal cardiomyocytes with siRNA led to upregulation of hypertrophic genes and increased leucine incorporation indicative of increased protein synthesis and hypertrophy. Finally, oral treatment of wild-type mice with a small molecule inhibitor of HIF-P4Hs phenocopied the effects of Hif-p4h-2 deficiency with less cardiomyocyte hypertrophy, upregulation of Hey2 and downregulation of the hypertrophy-associated genes. CONCLUSIONS: These results indicate that activation of the hypoxia response pathway upregulates Notch signaling and its target Hey2 resulting in transcriptional repression of hypertrophy-associated genes and less cardiomyocyte hypertrophy. This is eventually associated with better preserved cardiac function upon aging. Activation of the hypoxia response pathway thus has therapeutic potential for combating age-induced cardiac hypertrophy.
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Cardiomegalia , Hipoxia , Transducción de Señal , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cardiomegalia/genética , Cardiomegalia/metabolismo , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Ratones , RatasRESUMEN
[Figure: see text].
Asunto(s)
Aorta/enzimología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Técnicas de Inactivación de Genes , Hígado/enzimología , Placa Aterosclerótica , Prolil Hidroxilasas/genética , Animales , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Autoanticuerpos/sangre , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Lipoproteína Lipasa/metabolismo , Lipoproteínas LDL/inmunología , Lipoproteínas LDL/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Transcriptoma , Triglicéridos/sangreRESUMEN
The HIF prolyl 4-hydroxylases (HIF-P4H) control hypoxia-inducible factor (HIF), a powerful mechanism regulating cellular adaptation to decreased oxygenation. The gastrointestinal epithelium subsists in "physiological hypoxia" and should therefore have an especially well-designed control over this adaptation. Thus, we assessed the absolute mRNA expression levels of the HIF pathway components, Hif1a, HIF2a, Hif-p4h-1, 2 and 3 and factor inhibiting HIF (Fih1) in murine jejunum, caecum and colon epithelium using droplet digital PCR. We found a higher expression of all these genes towards the distal end of the gastrointestinal tract. We detected mRNA for Hif-p4h-1, 2 and 3 in all parts of the gastrointestinal tract. Hif-p4h-2 had significantly higher expression levels compared to Hif-p4h-1 and 3 in colon and caecum epithelium. To test the roles each HIF-P4H isoform plays in the gut epithelium, we measured the gene expression of classical HIF target genes in Hif-p4h-1-/-, Hif-p4h-2 hypomorph and Hif-p4h-3-/- mice. Only Hif-p4h-2 hypomorphism led to an upregulation of HIF target genes, confirming a predominant role of HIF-P4H-2. However, the abundance of Hif-p4h-1 and 3 expression in the gastrointestinal epithelium implies that these isoforms may have specific functions as well. Thus, the development of selective inhibitors might be useful for diverging therapeutic needs.
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Regulación Enzimológica de la Expresión Génica , Prolina Dioxigenasas del Factor Inducible por Hipoxia/genética , Hipoxia/enzimología , Hipoxia/genética , Mucosa Intestinal/enzimología , Envejecimiento/metabolismo , Animales , Ciego/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Isoenzimas/metabolismo , Yeyuno/metabolismo , Ratones Endogámicos C57BL , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de SeñalRESUMEN
Collagen is the most abundant protein in humans. It has a characteristic triple-helix structure and is heavily posttranslationally modified. The complex biosynthesis of collagen involves processing by many enzymes and chaperones in the rough endoplasmic reticulum. Lysyl hydroxylase 1 (LH1) is required to hydroxylate lysine for cross-linking and carbohydrate attachment within collagen triple helical sequences. Additionally, a recent study of prolyl 3-hydroxylase 3 (P3H3) demonstrated that this enzyme may be critical for LH1 activity; however, the details surrounding its involvement remain unclear. If P3H3 is an LH1 chaperone that is critical for LH1 activity, P3H3 and LH1 null mice should display a similar deficiency in lysyl hydroxylation. To test this hypothesis, we compared the amount and location of hydroxylysine in the triple helical domains of type V and I collagen from P3H3 null, LH1 null, and wild-type mice. The amount of hydroxylysine in type V collagen was reduced in P3H3 null mice, but surprisingly type V collagen from LH1 null mice contained as much hydroxylysine as type V collagen from wild-type mice. In type I collagen, our results indicate that LH1 plays a global enzymatic role in lysyl hydroxylation. P3H3 is also involved in lysyl hydroxylation, particularly at cross-link formation sites, but is not required for all lysyl hydroxylation sites. In summary, our study suggests that LH1 and P3H3 likely have two distinct mechanisms to recognize different collagen types and to distinguish cross-link formation sites from other sites in type I collagen.
Asunto(s)
Colágeno Tipo I/metabolismo , Colágeno Tipo V/metabolismo , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Colágeno/genética , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo V/genética , Retículo Endoplásmico Rugoso/metabolismo , Hidroxilación , Hidroxilisina/metabolismo , Lisina/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Procolágeno-Prolina Dioxigenasa/genética , Conformación Proteica , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
Collagens are the most abundant proteins in the extracellular matrix. They provide a framework to build organs and tissues and give structural support to make them resistant to mechanical load and forces. Several intra- and extracellular modifications are needed to make functional collagen molecules, intracellular post-translational modifications of proline and lysine residues having key roles in this. In this article, we provide a review on the enzymes responsible for the proline and lysine modifications, that is collagen prolyl 4-hydroxylases, 3-hydroxylases and lysyl hydroxylases, and discuss their biological functions and involvement in diseases.
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Colágeno/biosíntesis , Enfermedades del Tejido Conjuntivo/enzimología , Enfermedades del Tejido Conjuntivo/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Modelos Animales de Enfermedad , Glicosilación , Humanos , Hidroxilación , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Prolina Dioxigenasa/genéticaRESUMEN
Prolyl 4-hydroxylases (P4Hs) have vital roles in regulating collagen synthesis and hypoxia response. A transmembrane P4H (P4H-TM) is a recently identified member of the family. Biallelic loss of function P4H-TM mutations cause a severe autosomal recessive intellectual disability syndrome in humans, but functions of P4H-TM are essentially unknown at cellular level. Our microarray data on P4h-tm-/- mouse cortexes where P4H-TM is abundantly expressed indicated expression changes in genes involved in calcium signaling and expression of several calcium sequestering ATPases was upregulated in P4h-tm-/- primary mouse astrocytes. Cytosolic and intraorganellar calcium imaging of P4h-tm-/- cells revealed that receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) and calcium re-uptake by mitochondria were compromised. HIF1, but not HIF2, was found to be a key mediator of the P4H-TM effect on calcium signaling. Furthermore, total internal reflection fluorescence (TIRF) imaging showed that calcium agonist-induced gliotransmission was attenuated in P4h-tm-/- astrocytes. This phenotype was accompanied by redistribution of mitochondria from distal processes to central parts of the cell body and decreased intracellular ATP content. Our data show that P4H-TM is a novel regulator of calcium dynamics and gliotransmission.
Asunto(s)
Astrocitos , Señalización del Calcio , Astrocitos/metabolismo , Humanos , Hipoxia , Procolágeno-Prolina Dioxigenasa/metabolismo , Prolil HidroxilasasRESUMEN
Prolyl 4-hydroxylases (P4Hs) catalyze post-translational hydroxylation of peptidyl proline residues. In addition to collagen P4Hs and hypoxia-inducible factor P4Hs, a third P4H-the poorly characterized endoplasmic reticulum-localized transmembrane prolyl 4-hydroxylase (P4H-TM)-is found in animals. P4H-TM variants are associated with the familiar neurological HIDEA syndrome, but how these variants might contribute to disease is unknown. Here, we explored this question in a structural and functional analysis of soluble human P4H-TM. The crystal structure revealed an EF domain with two Ca2+-binding motifs inserted within the catalytic domain. A substrate-binding groove was formed between the EF domain and the conserved core of the catalytic domain. The proximity of the EF domain to the active site suggests that Ca2+ binding is relevant to the catalytic activity. Functional analysis demonstrated that Ca2+-binding affinity of P4H-TM is within the range of physiological Ca2+ concentration in the endoplasmic reticulum. P4H-TM was found both as a monomer and a dimer in the solution, but the monomer-dimer equilibrium was not regulated by Ca2+. The catalytic site contained bound Fe2+ and N-oxalylglycine, which is an analogue of the cosubstrate 2-oxoglutarate. Comparison with homologous P4H structures complexed with peptide substrates showed that the substrate-interacting residues and the lid structure that folds over the substrate are conserved in P4H-TM, whereas the extensive loop structures that surround the substrate-binding groove, generating a negative surface potential, are different. Analysis of the structure suggests that the HIDEA variants cause loss of P4H-TM function. In conclusion, P4H-TM shares key structural elements with other P4Hs while having a unique EF domain.
Asunto(s)
Dioxigenasas/química , Prolil Hidroxilasas/química , Cristalografía por Rayos X , Motivos EF Hand , Humanos , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
Cellular energy demands are met by uptake and metabolism of nutrients like glucose. The principal transcriptional regulator for adapting glycolytic flux and downstream pathways like de novo lipogenesis to glucose availability in many cell types is carbohydrate response element-binding protein (ChREBP). ChREBP is activated by glucose metabolites and post-translational modifications, inducing nuclear accumulation and regulation of target genes. Here we report that ChREBP is modified by proline hydroxylation at several residues. Proline hydroxylation targets both ectopically expressed ChREBP in cells and endogenous ChREBP in mouse liver. Functionally, we found that specific hydroxylated prolines were dispensable for protein stability but required for the adequate activation of ChREBP upon exposure to high glucose. Accordingly, ChREBP target gene expression was rescued by re-expressing WT but not ChREBP that lacks hydroxylated prolines in ChREBP-deleted hepatocytes. Thus, proline hydroxylation of ChREBP is a novel post-translational modification that may allow for therapeutic interference in metabolic diseases.
Asunto(s)
Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/biosíntesis , Regulación de la Expresión Génica , Glucosa/metabolismo , Hígado/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células HEK293 , Humanos , Hidroxilación , Masculino , Enfermedades Metabólicas/genética , Enfermedades Metabólicas/metabolismo , Ratones , Ratones Transgénicos , Prolina/genética , Prolina/metabolismoRESUMEN
The transcription factor Hypoxia-inducible factor 1 (HIF-1) plays a pivotal role in tissue regeneration. HIF-1 is negatively controlled by O2-dependent prolyl hydroxylases with a predominant role of prolyl hydroxylase 2 isoform (Phd2). Transgenic mice, hypomorphic for this isoform, accumulate more HIF-1 under normoxic conditions. Using these mice, we investigated the influence of Phd2 and HIF-1 on the regenerative capability of skeletal muscle tissue after myotrauma. Phd2-hypomorphic and wild type mice (on C57Bl/6 background) were grouped with regeneration times from 6 to 168 hours after closed mechanic muscle trauma to the hind limb. Tissue samples were analysed by immuno-staining and real-time PCR. Bone marrow derived macrophages of wild type and Phd2-hypomorphic mice were isolated and analysed via flow cytometry and quantitative real-time PCR. Phd2 reduction led to a higher regenerative capability due to enhanced activation of myogenic factors accompanied by induction of genes responsible for glucose and lactate metabolism in Phd2-hypomorphic mice. Macrophage infiltration into the trauma areas in hypomorphic mice started earlier and was more pronounced compared to wild type mice. Phd2-hypomorphic mice also showed higher numbers of macrophages in areas with sustained trauma 72 hours after myotrauma application. In conclusion, we postulate that the HIF-1 pathway is activated secondary to a Phd2 reduction which may lead to i) higher activation of myogenic factors, ii) increased number of positive stem cell proliferation markers, and iii) accelerated macrophage recruitment to areas of trauma, resulting in faster muscle tissue regeneration after myotrauma. With the current development of prolyl hydroxylase domain inhibitors, our findings point towards a potential clinical benefit after myotrauma.
Asunto(s)
Prolina Dioxigenasas del Factor Inducible por Hipoxia/deficiencia , Músculo Esquelético/fisiología , Regeneración/fisiología , Traumatismos de los Tejidos Blandos/fisiopatología , Animales , Proliferación Celular/fisiología , Modelos Animales de Enfermedad , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Prolina Dioxigenasas del Factor Inducible por Hipoxia/metabolismo , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Distribución Aleatoria , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Non-alcoholic fatty liver disease (NAFLD) parallels the global obesity epidemic with unmet therapeutic needs. We investigated whether inhibition of hypoxia-inducible factor prolyl 4-hydroxylase-2 (HIF-P4H-2), a key cellular oxygen sensor whose inhibition stabilizes HIF, would protect from NAFLD by subjecting HIF-P4H-2-deficient (Hif-p4h-2gt/gt) mice to a high-fat, high-fructose (HFHF) or high-fat, methionine-choline-deficient (HF-MCD) diet. On both diets, the Hif-p4h-2gt/gt mice gained less weight and had less white adipose tissue (WAT) and its inflammation, lower serum cholesterol levels, and lighter livers with less steatosis and lower serum ALT levels than the wild type (WT). The intake of fructose in majority of the Hif-p4h-2gt/gt tissues, including the liver, was 15-35% less than in the WT. We found upregulation of the key fructose transporter and metabolizing enzyme mRNAs, Slc2a2, Khka, and Khkc, and higher ketohexokinase activity in the Hif-p4h-2gt/gt small intestine relative to the WT, suggesting enhanced metabolism of fructose in the former. On the HF-MCD diet, the Hif-p4h-2gt/gt mice showed more browning of the WAT and increased thermogenesis. A pharmacological pan-HIF-P4H inhibitor protected WT mice on both diets against obesity, metabolic dysfunction, and liver damage. These data suggest that HIF-P4H-2 inhibition could be studied as a novel, comprehensive treatment strategy for NAFLD. KEY MESSAGES: ⢠HIF-P4H-2 inhibition enhances intestinal fructose metabolism protecting the liver. ⢠HIF-P4H-2 inhibition downregulates hepatic lipogenesis. ⢠Induced browning of WAT and increased thermogenesis can also mediate protection. ⢠HIF-P4H-2 inhibition offers a novel, comprehensive treatment strategy for NAFLD.