RÉSUMÉ
The Vps10p domain receptor SorCS2 is crucial for the development and function of the nervous system and essential for brain-derived neurotrophic factor (BDNF)-induced changes in neuronal morphology and plasticity. SorCS2 regulates the subcellular trafficking of the BDNF signaling receptor TrkB as well as selected neurotransmitter receptors in a manner that is dependent on the SorCS2 intracellular domain (ICD). However, the cellular machinery and adaptor protein (AP) interactions that regulate receptor trafficking via the SorCS2 ICD are unknown. We here identify four splice variants of human SorCS2 differing in the insertion of an acidic cluster motif and/or a serine residue within the ICD. We show that each variant undergoes posttranslational proteolytic processing into a one- or two-chain receptor, giving rise to eight protein isoforms, the expression of which differs between neuronal and nonneuronal tissues and is affected by cellular stressors. We found that the only variants without the serine were able to rescue BDNF-induced branching of SorCS2 knockout hippocampal neurons, while variants without the acidic cluster showed increased interactions with clathrin-associated APs AP-1, AP-2, and AP-3. Using yeast two-hybrid screens, we further discovered that all variants bound dynein light chain Tctex-type 3; however, only variants with an acidic cluster motif bound kinesin light chain 1. Accordingly, splice variants showed markedly different trafficking properties and localized to different subcellular compartments. Taken together, our findings demonstrate the existence of eight functional SorCS2 isoforms with differential capacity for interactions with cytosolic ligands dynein light chain Tctex-type 3 and kinesin light chain 1, which potentially allows cell-type specific SorCS2 trafficking and BDNF signaling.
Sujet(s)
Épissage alternatif , Système nerveux central , Récepteurs de surface cellulaire , Humains , Protéines adaptatrices de la transduction du signal/métabolisme , Épissage alternatif/physiologie , Facteur neurotrophique dérivé du cerveau/génétique , Facteur neurotrophique dérivé du cerveau/métabolisme , Dynéines/métabolisme , Kinésine/métabolisme , Liaison aux protéines , Isoformes de protéines/métabolisme , Récepteur trkB/métabolisme , Récepteurs de surface cellulaire/métabolisme , Système nerveux central/croissance et développement , Maturation post-traductionnelle des protéines , Transport des protéines/génétiqueRÉSUMÉ
Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.
Sujet(s)
Maladies cardiovasculaires , Syndrome néphrotique , Insuffisance rénale chronique , Humains , Souris , Animaux , Syndrome néphrotique/anatomopathologie , Proprotéine convertase 9/métabolisme , Protéine-2 apparentée au récepteur des LDL , Maladies cardiovasculaires/métabolisme , Protéinurie/génétique , Tubules contournés proximaux/anatomopathologie , Insuffisance rénale chronique/anatomopathologie , Souris knockout , Subtilisines/métabolismeRÉSUMÉ
Lowering blood cholesterol levels efficiently reduces the risk of developing atherosclerotic cardiovascular disease (ASCVD), including coronary artery disease (CAD), which is the main cause of death worldwide. CAD is caused by plaque formation, comprising cholesterol deposits in the coronary arteries. Proprotein convertase subtilisin kexin/type 9 (PCSK9) was discovered in the early 2000s and later identified as a key regulator of cholesterol metabolism. PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor in the liver, which is responsible for clearing LDL-cholesterol (LDL-C) from the circulation. Accordingly, gain-of-function PCSK9 mutations are causative of familial hypercholesterolemia, a severe condition with extremely high plasma cholesterol levels and increased ASCVD risk, whereas loss-of-function PCSK9 mutations are associated with very low LDL-C levels and protection against CAD. Since the discovery of PCSK9, extensive investigations in developing PCSK9 targeting therapies have been performed. The combined delineation of clear biology, genetic risk variants, and PCSK9 crystal structures have been major drivers in developing antagonistic molecules. Today, two antibody-based PCSK9 inhibitors have successfully progressed to clinical application and shown to be effective in reducing cholesterol levels and mitigating the risk of ASCVD events, including myocardial infarction, stroke, and death, without any major adverse effects. A third siRNA-based inhibitor has been FDA-approved but awaits cardiovascular outcome data. In this review, we outline the PCSK9 biology, focusing on the structure and nonsynonymous mutations reported in the PCSK9 gene and elaborate on PCSK9-lowering strategies under development. Finally, we discuss future perspectives with PCSK9 inhibition in other severe disorders beyond cardiovascular disease.
Sujet(s)
Anticholestérolémiants , Athérosclérose , Maladies cardiovasculaires , Maladie des artères coronaires , Hypercholestérolémie , Humains , Proprotéine convertase 9/génétique , Proprotéine convertase 9/métabolisme , Cholestérol LDL , Maladies cardiovasculaires/génétique , Maladies cardiovasculaires/prévention et contrôle , Maladies cardiovasculaires/traitement médicamenteux , Hypercholestérolémie/traitement médicamenteux , Maladie des artères coronaires/traitement médicamenteux , Athérosclérose/traitement médicamenteux , Anticholestérolémiants/usage thérapeutiqueRÉSUMÉ
PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo. Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.
RÉSUMÉ
The multifunctional type 1 receptor sortilin is involved in endocytosis and intracellular transport of ligands. The short intracellular domain of sortilin binds several cytoplasmic adaptor proteins (e.g., the AP-1 complex and GGA1 to -3), most of which target two well-defined motifs: a C-terminal acidic cluster dileucine motif and a YXXΦ motif in the proximal third of the domain. Both motifs contribute to endocytosis as well as Golgi-endosome trafficking of sortilin. The C-terminal acidic cluster harbors a serine residue, which is subject to phosphorylation by casein kinase. Phosphorylation of this serine residue is known to modulate adaptor binding to sortilin. Here, we show that the cytoplasmic domain of sortilin also engages Rac-p21-activated kinases 1 to 3 (PAK1-3) via a binding segment that includes a tyrosine-based motif, also encompassing a serine residue. We further demonstrate that PAK1-3 specifically phosphorylate this serine residue and that this phosphorylation alters the affinity for AP-1 binding and consequently changes the intracellular localization of sortilin as a result of modulated trafficking. Our findings suggest that trafficking of ligands bound to sortilin is in part regulated by group A PAK kinases, which are downstream effectors of Rho GTPases and are known to affect a variety of processes by remodeling the cytoskeleton and by promoting gene transcription and cell survival.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/métabolisme , p21-Activated Kinases/métabolisme , Protéines adaptatrices du transport vésiculaire/analyse , Animaux , Cellules CHO , Cellules cultivées , Cricetulus , Cellules HEK293 , Humains , Souris de lignée C57BL , Phosphorylation , Domaines protéiques , Transport des protéinesRÉSUMÉ
BACKGROUND: Chronic kidney disease is a risk factor for premature development of coronary atherosclerosis and mortality. A high level of proprotein convertase subtilisin/kexin type 9 (PCSK9) is a recently recognized cardiovascular risk factor and has become the target of effective inhibitory treatment. In 167 kidney transplantation candidates, we aimed to: (i) compare levels of PCSK9 with those of healthy controls, (ii) examine the association between levels of PCSK9 and low-density lipoprotein cholesterol (LDL-c) and the degree of coronary artery disease (CAD) and (iii) evaluate if levels of PCSK9 predict major adverse cardiac events (MACE) and mortality. METHODS: Kidney transplant candidates (n = 167) underwent coronary computed tomography angiography (CCTA) and invasive coronary angiography (ICA) before transplantation. MACE and mortality data were extracted from the Western Denmark Heart Registry, a review of patient records and patient interviews. A group of 79 healthy subjects were used as controls. RESULTS: Mean PCSK9 levels did not differ between healthy controls and kidney transplant candidates. In patients not receiving lipid-lowering therapy, PCSK9 correlated positively with LDL-c (rho = 0.24, P < 0.05). Mean PCSK9 was similar in patients with and without obstructive CAD at both CCTA and ICA. In a multiple regression analysis, PCSK9 was associated with neither LDL-c (ß=-6.45, P = 0.44) nor coronary artery calcium score (ß=2.17, P = 0.84). During a follow-up of 3.7 years, PCSK9 levels were not associated with either MACE or mortality. CONCLUSIONS: The ability of PCSK9 levels to predict cardiovascular disease and prognosis does not seem to apply to a cohort of kidney transplant candidates.
Sujet(s)
Marqueurs biologiques/sang , Maladies cardiovasculaires/diagnostic , Cholestérol LDL/sang , Proprotéine convertase 9/sang , Insuffisance rénale chronique/complications , Maladies cardiovasculaires/sang , Maladies cardiovasculaires/étiologie , Études cas-témoins , Femelle , Humains , Mâle , Adulte d'âge moyen , Pronostic , Études prospectives , Insuffisance rénale chronique/sang , Insuffisance rénale chronique/anatomopathologie , Facteurs de risqueRÉSUMÉ
SorLA and Sortilin are multifunctional receptors involved in endocytosis and intracellular sorting of different and unrelated ligands. SorLA has recently attracted much attention as a novel strong risk gene for Alzheimer's disease, and much effort is currently being put into understanding the underlying molecular mechanism. Trafficking of SorLA and Sortilin are mediated by interacting with AP-1, AP-2, GGA 1-3 and the retromer complex. Although these cytosolic adaptor proteins all bind to both SorLA and Sortilin, a large fraction of intracellular Sortilin and SorLA are located in different subcellular vesicles. This indicates that unknown specialised adaptor proteins targeting SorLA for trafficking are yet to be discovered. We have identified HSPA12A as a new adaptor protein that, among Vps10p-D receptors, selectively binds to SorLA in an ADP/ATP dependent manner. This is the first described substrate of HSPA12A, and we demonstrate that the binding, which affects both endocytic speed and subcellular localisation of SorLA, is mediated by specific acidic residues in the cytosolic domain of SorLA. The identification of the relatively unknown HSPA12A as a SorLA specific interaction partner could lead to novel insight into the molecular mechanism of SorLA, and re-emphasises the role of heat shock proteins in neurodegenerative diseases.
Sujet(s)
Protéines du choc thermique HSP70/métabolisme , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Protéines adaptatrices du transport vésiculaire/composition chimique , Protéines adaptatrices du transport vésiculaire/métabolisme , Séquence d'acides aminés , Animaux , Astrocytes/cytologie , Astrocytes/métabolisme , Cellules HEK293 , Protéines du choc thermique HSP70/composition chimique , Humains , Protéines apparentées au récepteur LDL/composition chimique , Protéines de transport membranaire/composition chimique , Souris , Souris de lignée C57BL , Liaison aux protéines , Domaines protéiques , Transport des protéines , Spécificité du substrat , Techniques de double hybrideRÉSUMÉ
Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.PCSK9 interacts with LDL receptor, causing its degradation, and consequently reduces the clearance of LDL. Here, Gustafsen et al. show that PCSK9 interacts with heparan sulfate proteoglycans and this binding favors LDLR degradation. Pharmacological inhibition of this binding can be exploited as therapeutic intervention to lower LDL levels.
Sujet(s)
Protéoglycanes à sulfate d'héparane/métabolisme , Proprotéine convertase 9/métabolisme , Récepteurs aux lipoprotéines LDL/métabolisme , Anticorps/pharmacologie , Sites de fixation , Antienzymes/pharmacologie , Cellules HepG2 , Héparine/composition chimique , Héparine/pharmacologie , Hépatocytes/métabolisme , Humains , Inhibiteurs de PCSK9 , Proprotéine convertase 9/composition chimique , Proprotéine convertase 9/génétique , ProtéolyseRÉSUMÉ
SorLA is a neuronal sorting receptor that is genetically associated with Alzheimer disease. SorLA interacts directly with the amyloid precursor protein (APP) and affects the processing of the precursor, leading to a decreased generation of the amyloid-ß peptide. The SorLA complement-type repeat (CR) domains associate in vitro with APP, but the precise molecular determinants of SorLA·APP complex formation and the mechanisms responsible for the effect of binding on APP processing have not yet been elucidated. Here, we have generated protein expression constructs for SorLA devoid of the 11 CR-domains and for two SorLA mutants harboring substitutions of the fingerprint residues in the central CR-domains. We generated SH-SY5Y cell lines that stably express these SorLA variants to study the binding and processing of APP using co-immunoprecipitation and Western blotting/ELISAs, respectively. We found that the SorLA CR-cluster is essential for interaction with APP and that deletion of the CR-cluster abolishes the protection against APP processing. Mutation of identified fingerprint residues in the SorLA CR-domains leads to changes in the O-linked glycosylation of APP when expressed in SH-SY5Y cells. Our results provide novel information on the mechanisms behind the influence of SorLA activity on APP metabolism by controlling post-translational glycosylation in the Golgi, suggesting new strategies against amyloidogenesis in Alzheimer disease.
Sujet(s)
Précurseur de la protéine bêta-amyloïde/métabolisme , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Maturation post-traductionnelle des protéines , Sites de fixation , Lignée cellulaire tumorale , Glycosylation , Humains , Protéines apparentées au récepteur LDL/composition chimique , Protéines apparentées au récepteur LDL/génétique , Protéines de transport membranaire/composition chimique , Protéines de transport membranaire/génétique , Liaison aux protéines , Transport des protéinesRÉSUMÉ
Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75(NTR). CNS neurons, but not glia, express SorCS2 as a single-chain protein that is essential for proBDNF-induced growth cone collapse in developing dopaminergic processes. SorCS2- or p75(NTR)-deficient in mice caused reduced dopamine levels and metabolism and dopaminergic hyperinnervation of the frontal cortex. Accordingly, both knockout models displayed a paradoxical behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75(NTR)-positive dying Schwann cells, with apoptosis being profoundly attenuated in Sorcs2(-/-) mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins.
Sujet(s)
Apoptose , Encéphale/métabolisme , Neurones dopaminergiques/métabolisme , Réseau nerveux/métabolisme , Protéines de tissu nerveux/composition chimique , Protéines de tissu nerveux/métabolisme , Récepteurs de surface cellulaire/composition chimique , Récepteurs de surface cellulaire/métabolisme , Cellules de Schwann/métabolisme , Animaux , Encéphale/embryologie , Facteur neurotrophique dérivé du cerveau/métabolisme , Corps strié/composition chimique , Dopamine/analyse , Dopamine/métabolisme , Lobe frontal/composition chimique , Cônes de croissance/métabolisme , Cellules HEK293 , Humains , Mâle , Souris , Souris de lignée C57BL , Souris knockout , Neurones/métabolisme , Récepteurs facteur croissance nerf/métabolisme , Substantia nigra/métabolisme , Aire tegmentale ventrale/métabolismeRÉSUMÉ
Circulating PCSK9 destines low-density lipoprotein receptor for degradation in lysosomes, resulting in increased LDL cholesterol. Accordingly, it is an attractive drug target for hypercholesterolemia, and results from clinical trials are promising. While the physiological role of PCSK9 in cholesterol metabolism is well described, its complex mechanism of action remains poorly understood, although it is known to depend on intracellular trafficking. We here identify sortilin, encoded by the hypercholesterolemia-risk gene SORT1, as a high-affinity sorting receptor for PCSK9. Sortilin colocalizes with PCSK9 in the trans-Golgi network and facilitates its secretion from primary hepatocytes. Accordingly, sortilin-deficient mice display decreased levels of circulating PCSK9, while sortilin overexpression in the liver confers increased plasma PCSK9. Furthermore, circulating PCSK9 and sortilin were positively correlated in a human cohort of healthy individuals, suggesting that sortilin is involved in PCSK9 secretion in humans. Taken together, our findings establish sortilin as a critical regulator of PCSK9 activity.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/sang , Hypercholestérolémie/sang , Proprotein convertases/sang , Serine endopeptidases/sang , Protéines adaptatrices du transport vésiculaire/métabolisme , Animaux , Lignée cellulaire , Technique d'immunofluorescence , Humains , Hypercholestérolémie/métabolisme , Souris , Souris knockout , Modèles biologiques , Proprotéine convertase 9 , Proprotein convertases/métabolisme , Liaison aux protéines , Serine endopeptidases/métabolismeRÉSUMÉ
Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson's disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.
Sujet(s)
Récepteurs des facteurs neurotrophiques dérivés des cellules gliales/métabolisme , Facteur neurotrophique dérivé des cellules gliales/métabolisme , Protéines de transport membranaire/métabolisme , Protéines proto-oncogènes c-ret/métabolisme , Récepteurs aux lipoprotéines LDL/métabolisme , Animaux , Anxiété/métabolisme , Anxiété/anatomopathologie , Comportement animal , Membrane cellulaire/métabolisme , Survie cellulaire , Neurones dopaminergiques/cytologie , Neurones dopaminergiques/métabolisme , Endocytose , Cellules HEK293 , Humains , Lysosomes/métabolisme , Protéines de transport membranaire/déficit , Souris , Souris de lignée C57BL , Souris knockout , Complexes multiprotéiques/métabolisme , Liaison aux protéines , Transport des protéines , Récepteurs aux lipoprotéines LDL/déficitRÉSUMÉ
The development and progression of Alzheimer's disease is linked to excessive production of toxic amyloid-ß peptide, initiated by ß-secretase cleavage of the amyloid precursor protein (APP). In contrast, soluble APPα (sAPPα) generated by the α-secretase is known to stimulate dendritic branching and enhance synaptic function. Regulation of APP processing, and the shift from neurotrophic to neurotoxic APP metabolism remains poorly understood, but the cellular localization of APP and its interaction with various receptors is considered important. We here identify sortilin as a novel APP interaction partner. Like the related APP receptor SorLA, sortilin is highly expressed in the CNS, but whereas SorLA mainly colocalizes with APP in the soma, sortilin interacts with APP in neurites. The presence of sortilin promotes α-secretase cleavage of APP, unlike SorLA, which inhibits the generation of all soluble products. Also, sortilin and SorLA both bind and mediate internalization of sAPP but to different cellular compartments. The interaction involves the 6A domain of APP, present in both neuronal and non-neuronal APP isoforms. This is important as sAPP receptors described so far only bind the non-neuronal isoforms, leaving SorLA and sortilin as the only receptors for sAPP generated by neurons. Together, our findings establish sortilin, as a novel APP interaction partner that influences both production and cellular uptake of sAPP.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/métabolisme , Précurseur de la protéine bêta-amyloïde/métabolisme , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Neurones/métabolisme , Animaux , Cellules CHO , Cricetinae , Cellules HEK293 , Humains , Neurites/métabolisme , Transport des protéines/physiologieRÉSUMÉ
Apolipoprotein E (APOE) is the major risk factor for sporadic Alzheimer's disease. Among other functions, APOE is proposed to sequester neurotoxic amyloid-ß (Aß) peptides in the brain, delivering them to cellular catabolism via neuronal APOE receptors. Still, the receptors involved in this process remain controversial. Here, we identified the pro-neurotrophin receptor sortilin as major endocytic pathway for clearance of APOE/Aß complexes in neurons. Sortilin binds APOE with high affinity. Lack of receptor expression in mice results in accumulation of APOE and of Aß in the brain and in aggravated plaque burden. Also, primary neurons lacking sortilin exhibit significantly impaired uptake of APOE/Aß complexes despite proper expression of other APOE receptors. Despite higher than normal brain APOE levels, sortilin-deficient animals display anomalies in brain lipid metabolism (e.g., accumulation of sulfatides) seen in APOE-deficient mice, indicating functional deficiency in cellular APOE uptake pathways. Together, our findings identified sortilin as an essential neuronal pathway for APOE-containing lipoproteins in vivo and suggest an intriguing link between Aß catabolism and pro-neurotrophin signaling converging on this receptor.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/métabolisme , Peptides bêta-amyloïdes/métabolisme , Encéphale/métabolisme , Protéine-1 apparentée au récepteur des LDL/métabolisme , Neurones/métabolisme , Animaux , Apolipoprotéines E/métabolisme , Astrocytes/métabolisme , Souris , Plaque amyloïde/métabolismeRÉSUMÉ
sorLA is a sorting receptor for amyloid precursor protein (APP) genetically linked to Alzheimer's disease (AD). Retromer, an adaptor complex in the endosome-to-Golgi retrieval pathway, has been implicated in APP transport because retromer deficiency leads to aberrant APP sorting and processing and levels of retromer proteins are altered in AD. Here we report that sorLA and retromer functionally interact in neurons to control trafficking and amyloidogenic processing of APP. We have identified a sequence (FANSHY) in the cytoplasmic domain of sorLA that is recognized by the VPS26 subunit of the retromer complex. Accordingly, we characterized the interaction between the retromer complex and sorLA and determined the role of retromer on sorLA-dependent sorting and processing of APP. Mutations in the VPS26 binding site resulted in receptor redistribution to the endosomal network, similar to the situation seen in cells with VPS26 knockdown. The sorLA mutant retained APP-binding activity but, as opposed to the wild-type receptor, misdirected APP into a distinct non-Golgi compartment, resulting in increased amyloid processing. In conclusion, our data provide a molecular link between reduced retromer expression and increased amyloidogenesis as seen in patients with sporadic AD.
Sujet(s)
Précurseur de la protéine bêta-amyloïde/métabolisme , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Modification traductionnelle des protéines/physiologie , Motifs d'acides aminés/génétique , Séquence d'acides aminés , Précurseur de la protéine bêta-amyloïde/génétique , Animaux , Humains , Protéines apparentées au récepteur LDL/génétique , Protéines de transport membranaire/génétique , Données de séquences moléculaires , Cellules PC12 , Liaison aux protéines/génétique , Motifs et domaines d'intéraction protéique/génétique , Modification traductionnelle des protéines/génétique , Transport des protéines/génétique , RatsRÉSUMÉ
The precursor of the neurotrophin (NT) nerve growth factor (NGF) (proNGF) serves physiological functions distinct from its mature counterpart as it induces neuronal apoptosis through activation of a p75 NT receptor (p75(NTR) ) and Sortilin death-signalling complex. The NTs brain-derived nerve growth factor (BDNF) and NT3 provide essential trophic support to auditory neurons. Injury to the NT-secreting cells in the inner ear is followed by irreversible degeneration of spiral ganglion neurons with consequences such as impaired hearing or deafness. Lack of mature NTs may explain the degeneration of spiral ganglion neurons, but another mechanism is possible as unprocessed proNTs released from the injured cells may contribute to the degeneration by induction of apoptosis. Recent studies demonstrate that proBDNF, like proNGF, is a potent inducer of Sortilin:p75(NTR) -mediated apoptosis. In addition, a coincident upregulation of proBDNF and p75(NTR) has been observed in degenerating spiral ganglion neurons, but the Sortilin expression in the inner ear is unresolved. Here we demonstrate that Sortilin and p75(NTR) are coexpressed in neurons of the neonatal inner ear. Furthermore, we establish that proNT3 exhibits high-affinity binding to Sortilin and has the capacity to enhance cell surface Sortilin:p75(NTR) complex formation as well as to mediate apoptosis in neurons coexpressing p75(NTR) and Sortilin. Based on the examination of wildtype and Sortilin-deficient mouse embryos, Sortilin does not significantly influence the developmental selection of spiral ganglion neurons. However, our results suggest that proNT3 and proBDNF may play important roles in the response to noise-induced injuries or ototoxic damage via the Sortilin:p75(NTR) death-signalling complex.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/métabolisme , Apoptose/physiologie , Oreille interne/cytologie , Neurones/physiologie , Neurotrophine-3/métabolisme , Précurseurs de protéines/métabolisme , Protéines adaptatrices du transport vésiculaire/génétique , Animaux , Facteur neurotrophique dérivé du cerveau/génétique , Facteur neurotrophique dérivé du cerveau/métabolisme , Cellules cultivées , Cellules HEK293 , Humains , Souris , Souris de lignée C57BL , Complexes multiprotéiques/métabolisme , Neurones/cytologie , Neurotrophine-3/génétique , Liaison aux protéines , Précurseurs de protéines/génétique , Rats , Rat Wistar , Récepteur facteur croissance nerf/génétique , Récepteur facteur croissance nerf/métabolisme , Ganglion spiral/cytologieRÉSUMÉ
The cytosolic adaptors GGA1-3 mediate sorting of transmembrane proteins displaying a C-terminal acidic dileucine motif (DXXLL) in their cytosolic domain. GGA1 and GGA3 contain similar but intrinsic motifs that are believed to serve as autoinhibitory sites activated by the phosphorylation of a serine positioned three residues upstream of the DXXLL motif. In the present study, we have subjected the widely acknowledged concept of GGA1 autoinhibition to a thorough structural and functional examination. We find that (i) the intrinsic motif of GGA1 is inactive, (ii) only C-terminal DXXLL motifs constitute active GGA binding sites, (iii) while aspartates and phosphorylated serines one or two positions upstream of the DXXLL motif increase GGA1 binding, phosphoserines further upstream have little or no influence and (iv) phosphorylation of GGA1 does not affect its conformation or binding to Sortilin and SorLA. Taken together, our findings seem to refute the functional significance of GGA autoinhibition in particular and of intrinsic GGA binding motifs in general.
Sujet(s)
Protéines adaptatrices du transport vésiculaire/antagonistes et inhibiteurs , Protéines adaptatrices du transport vésiculaire/métabolisme , Protéines adaptatrices du transport vésiculaire/composition chimique , Protéines adaptatrices du transport vésiculaire/génétique , Motifs d'acides aminés , Séquence d'acides aminés , Lignée cellulaire , Cristallographie aux rayons X , Humains , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Modèles moléculaires , Données de séquences moléculaires , Mutation/génétique , Phosphosérine , Liaison aux protéines , Motifs et domaines d'intéraction protéique , Signaux de triage des protéines , Saccharomyces cerevisiae/génétique , Techniques de double hybrideRÉSUMÉ
The type I transmembrane protein SorCS1 is a member of the Vps10p-domain receptor family comprised of Sortilin, SorLA and SorCS1, -2 and -3. Current information indicates that Sortilin and SorLA mediate intracellular protein trafficking and sorting, but little is known about the cellular functions of the SorCS subgroup. SorCS1 binds platelet-derived growth factor-BB (PDGF-BB) and is expressed in isoforms differing only in their cytoplasmic domains. Here, we identify two novel isoforms of mouse SorCS1 designated m-SorCS1c and -d. In situ hybridization revealed a combinatorial expression pattern of the variants in brain and embryonic tissues. We demonstrate that among the mouse variants, only SorCS1c mediates internalization and that the highly conserved SorCS1c is internalized through a canonical tyrosine-based motif. In contrast, human SorCS1a, whose cytoplasmic domain is completely different from mouse SorCS1a, is internalized through a DXXLL motif. We report that the human SorCS1a cytoplasmic domain interacts with the alphaC/sigma2 subunits of the adaptor protein (AP)-2 complex, and internalization of human SorCS1a and -c is mediated by AP-2. Our results suggest that the endocytic isoforms target internalized cargo to lysosomes but are not engaged in Golgi-endosomal transport to a significant degree.
Sujet(s)
Glycoprotéines membranaires/composition chimique , Glycoprotéines membranaires/métabolisme , Protéines de transport membranaire/composition chimique , Protéines de transport membranaire/métabolisme , Récepteurs de surface cellulaire/composition chimique , Récepteurs de surface cellulaire/métabolisme , Motifs d'acides aminés , Séquence d'acides aminés , Animaux , Animaux nouveau-nés , Cellules CHO , Cricetinae , Cricetulus , Fibroblastes/métabolisme , Humains , Immunohistochimie , Hybridation in situ , Glycoprotéines membranaires/génétique , Protéines de transport membranaire/génétique , Souris , Isoformes de protéines/composition chimique , Structure tertiaire des protéines , Transport des protéines , Récepteurs de surface cellulaire/génétique , Analyse de séquence de protéine , Distribution tissulaireRÉSUMÉ
SorLA/LR11 (250 kDa) is the largest and most composite member of the Vps10p-domain receptors, a family of type 1 proteins preferentially expressed in neuronal tissue. SorLA binds several ligands, including neurotensin, platelet-derived growth factor-bb, and lipoprotein lipase, and via complex-formation with the amyloid precursor protein it downregulates generation of Alzheimer's disease-associated Abeta-peptide. The receptor is mainly located in vesicles, suggesting a function in protein sorting and transport. Here we examined SorLA's trafficking using full-length and chimeric receptors and find that its cytoplasmic tail mediates efficient Golgi body-endosome transport, as well as AP-2 complex-dependent endocytosis. Functional sorting sites were mapped to an acidic cluster-dileucine-like motif and to a GGA binding site in the C terminus. Experiments in permanently or transiently AP-1 mu1-chain-deficient cells established that the AP-1 adaptor complex is essential to SorLA's transport between Golgi membranes and endosomes. Our results further implicate the GGA proteins in SorLA trafficking and provide evidence that SNX1 and Vps35, as parts of the retromer complex or possibly in a separate context, are engaged in retraction of the receptor from endosomes.
Sujet(s)
Précurseur de la protéine bêta-amyloïde/métabolisme , Protéines apparentées au récepteur LDL/métabolisme , Protéines de transport membranaire/métabolisme , Transport des protéines , Protéines de fusion recombinantes/métabolisme , Protéines adaptatrices du transport vésiculaire , Séquence d'acides aminés , Animaux , Lignée cellulaire , Cricetinae , Cricetulus , Endosomes/métabolisme , Appareil de Golgi/métabolisme , Humains , Protéines apparentées au récepteur LDL/génétique , Glycoprotéines membranaires/génétique , Glycoprotéines membranaires/métabolisme , Protéines de transport membranaire/génétique , Souris , Données de séquences moléculaires , Protéines de tissu nerveux/génétique , Protéines de tissu nerveux/métabolisme , Structure tertiaire des protéines , Interférence par ARN , Récepteurs à l'interleukine-2/génétique , Récepteurs à l'interleukine-2/métabolisme , Protéines de fusion recombinantes/génétique , Techniques de double hybrideRÉSUMÉ
To elucidate the function of the ndx homeobox genes during the Rhizobium-legume symbiosis, two Lotus japonicus ndr genes were expressed in the antisense orientation under the control of the nodule-expressed promoter Psenod12 in transgenic Lotus japonicus plants. Many of the transformants obtained segregated into plants that failed to sustain proper development and maintenance of root nodules concomitant with down-regulation of the two ndx genes. The root nodules were actively fixing nitrogen 3 weeks after inoculation, but the plants exhibited a stunted growth phenotype. The nodules on such antisense plants had under-developed vasculature and lenticels when grown on medium lacking nitrogen sources. These nodules furthermore entered senescence earlier than the wild-type nodules. Normal plant growth was resumed upon external addition of nitrogen. This suggests that assimilated nitrogen is not properly supplied to the plants in which the two ndx genes are down-regulated. The results presented here, indicate that the ndx genes play a role in the development of structural nodule features, required for proper gas diffusion into the nodule and/or transport of the assimilated nitrogen to the plant.