Susana Granell

Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance.

Vinblastine and other microtubule inhibitors used as antimitotic cancer drugs characteristically promote the phosphorylation of the key anti-apoptotic protein, Bcl-xL. However, putative sites of phosphorylation have been inferred based on potential recognition by JNK, and no direct biochemical analysis has been performed. In this study we used protein purification and mass spectrometry to identify Ser-62 as a single major site in vivo. Site-directed mutagenesis confirmed Ser-62 to be the site of Bcl-xL phosphorylation induced by several microtubule inhibitors tested. Vinblastine-treated cells overexpressing a Ser-62 → Ala mutant showed highly significantly reduced apoptosis compared with cells expressing wild-type Bcl-xL. Co-immunoprecipitation revealed that phosphorylation caused wild-type Bcl-xL to release bound Bax, whereas phospho-defective Bcl-xL retained the ability to bind Bax. In contrast, phospho-mimic (Ser-62 → Asp) Bcl-xL exhibited a reduced capacity to bind Bax. Functional tests were performed by transiently co-transfecting Bax in the context of different Bcl-xL mutants. Co-expression of wild-type or phospho-defective Bcl-xL counteracted the adverse effects of Bax expression on cell viability, whereas phospho-mimic Bcl-xL failed to provide the same level of protection against Bax. These studies suggest that Bcl-xL phosphorylation induced by microtubule inhibitors plays a key pro-apoptotic role at least in part by disabling the ability of Bcl-xL to bind Bax.

Constitutive Traffic of Melanocortin-4 Receptor in Neuro2A Cells and Immortalized Hypothalamic Neurons

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor (GPCR) that binds α-melanocyte-stimulating hormone (α-MSH) and has a central role in the regulation of appetite and energy expenditure. Most GPCRs are endocytosed following binding to the agonist and receptor desensitization. Other GPCRs are internalized and recycled back to the plasma membrane constitutively, in the absence of the agonist. In unstimulated neuroblastoma cells and immortalized hypothalamic neurons, epitopetagged MC4R was localized both at the plasma membrane and in an intracellular compartment. These two pools of receptors were in dynamic equilibrium, with MC4R being rapidly internalized and exocytosed. In the absence of α-MSH, a fraction of cell surface MC4R localized together with transferrin receptor and to clathrin-coated pits. Constitutive MC4R internalization was impaired by expression of a dominant negative dynamin mutant. Thus, MC4R is internalized together with transferrin receptor by clathrin-dependent endocytosis. Cell exposure toα-MSH reduced the amount of MC4R at the plasma membrane by blocking recycling of a fraction of internalized receptor, rather than by increasing its rate of endocytosis. The data indicate that, in neuronal cells, MC4R recycles constitutively and that α-MSH modulates MC4R residency at the plasma membrane by acting at an intracellular sorting step.

Obesity-linked variants of melanocortin-4 receptor are misfolded in the endoplasmic reticulum and can be rescued to the cell surface by a chemical chaperone.

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find that these variants are all retained in the endoplasmic reticulum (ER), are ubiquitinated to a greater extent than the wild-type (wt) receptor, and induce ER stress with increased levels of ER chaperones as compared with wt-MC4R and appearance of CCAAT/enhancer-binding protein homologous protein (CHOP). Expression of the X-box-binding-protein-1 (XBP-1) with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone 4-phenyl butyric acid (PBA) increased the cell surface expression of wt-MC4R, MC4R-I316S, and I317T by more than 40%. PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functions to promote MC4R folding. MC4R-I316S rescued to the cell surface is functional, with a 52% increase in agonist-induced cAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor of the ubiquitin-activating enzyme 1 increased by approximately 40% the expression of the receptors at the cell surface, and the effects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.

Activating Transcription Factor 6 Limits Intracellular Accumulation of Mutant α1-Antitrypsin Z and Mitochondrial Damage in Hepatoma Cells

Background: A variant of α1-antitrypsin with an E342K mutation (ATZ) is retained in the endoplasmic reticulum (ER) of hepatocytes. Results: In hepatoma cells, activation of a branch of the unfolded protein response (UPR) increases ER-dependent degradation of ATZ. Conclusion: ATZ degradation can be accelerated by induction of a protective branch of the UPR. Significance: UPR-dependent ATZ disposal is a potential target for therapy. α1-Antitrypsin is a serine protease inhibitor secreted by hepatocytes. A variant of α1-antitrypsin with an E342K (Z) mutation (ATZ) has propensity to form polymers, is retained in the endoplasmic reticulum (ER), is degraded by both ER-associated degradation and autophagy, and causes hepatocyte loss. Constant features in hepatocytes of PiZZ individuals and in PiZ transgenic mice expressing ATZ are the formation of membrane-limited globular inclusions containing ATZ and mitochondrial damage. Expression of ATZ in the liver does not induce the unfolded protein response (UPR), a protective mechanism aimed to maintain ER homeostasis in the face of an increased load of proteins. Here we found that in hepatoma cells the ER E3 ligase HRD1 functioned to degrade most of the ATZ before globular inclusions are formed. Activation of the activating transcription factor 6 (ATF6) branch of the UPR by expression of spliced ATF6(1–373) decreased intracellular accumulation of ATZ and the formation of globular inclusions by a pathway that required HRD1 and the proteasome. Expression of ATF6(1–373) in ATZ-expressing hepatoma cells did not induce autophagy and increased the level of the proapoptotic factor CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) but did not lead to apoptotic DNA fragmentation. Expression of ATF6(1–373) did not cause inhibition of protein synthesis and prevented mitochondrial damage induced by ATZ expression. It was concluded that activation of the ATF6 pathway of the UPR limits ATZ-dependent cell toxicity by selectively promoting ER-associated degradation of ATZ and is thereby a potential target to prevent hepatocyte loss in addition to autophagy-enhancing drugs.

Constitutive Cholesterol-dependent Endocytosis of Melanocortin-4 Receptor (MC4R) Is Essential to Maintain Receptor Responsiveness to α-Melanocyte-stimulating Hormone (α-MSH)

Background: MC4R is essential for energy homeostasis and cycles continuously. Results: MC4R internalization is blocked by clathrin and cholesterol depletion, reducing receptor response to α-MSH, which is partially recovered by mutations at Thr-312/Ser-329. Conclusion: Constitutive internalization of MC4R is cholesterol-dependent and required for receptor function. Significance: These findings provide a potentially novel mechanism by which hypothalamic cell cholesterol content can affect appetite control. Melanocortin-4 receptor (MC4R) is a G-protein-coupled receptor expressed in the hypothalamus where it controls feeding behavior. MC4R cycles constitutively and is internalized at the same rate in the presence or absence of stimulation by the agonist, melanocyte-stimulating hormone (α-MSH). This is different from other G-protein-coupled receptors, such as β2-adrenergic receptor (β2AR), which internalizes more rapidly in response to agonist stimulation. Here, it is found that in immortalized neuronal Neuro2A cells expressing exogenous receptors, constitutive endocytosis of MC4R and agonist-dependent internalization of β2AR were equally sensitive to clathrin depletion. Inhibition of MC4R endocytosis by clathrin depletion decreased the number of receptors at the cell surface that were responsive to the agonist, α-MSH, by 75%. Mild membrane cholesterol depletion also inhibited constitutive endocytosis of MC4R by ∼5-fold, while not affecting recycling of MC4R or agonist-dependent internalization of β2AR. Reduced cholesterol did not change the MC4R dose-response curve to α-MSH, but it decreased the amount of cAMP generated per receptor number indicating that a population of MC4R at the cell surface becomes nonfunctional. The loss of MC4R function increased over time (25–50%) and was partially reversed by mutations at putative phosphorylation sites (T312A and S329A). This was reproduced in hypothalamic GT1-7 cells expressing endogenous MC4R. The data indicate that constitutive endocytosis of MC4R is clathrin- and cholesterol-dependent. MC4R endocytosis is required to maintain MC4R responsiveness to α-MSH by constantly eliminating from the plasma membrane a pool of receptors modified at Thr-312 and Ser-329 that have to be cycled to the endosomal compartment to regain function.

A Novel Melanocortin-4 Receptor Mutation MC4R-P272L Associated with Severe Obesity Has Increased Propensity To Be Ubiquitinated in the ER in the Face of Correct Folding

Heterozygous mutations in the melanocortin-4 receptor (MC4R) gene represent the most frequent cause of monogenic obesity in humans. MC4R mutation analysis in a cohort of 77 children with morbid obesity identified previously unreported heterozygous mutations (P272L, N74I) in two patients inherited from their obese mothers. A rare polymorphism (I251L, allelic frequency: 1/100) reported to protect against obesity was found in another obese patient. When expressed in neuronal cells, the cell surface abundance of wild-type MC4R and of the N74I and I251L variants and the cAMP generated by these receptors in response to exposure to the agonist, α-MSH, were not different. Conversely, MC4R P272L was retained in the endoplasmic reticulum and had reduced cell surface expression and signaling (by ≈3-fold). The chemical chaperone PBA, which promotes protein folding of wild-type MC4R, had minimal effects on the distribution and signaling of the P272L variant. In contrast, incubation with UBE-41, a specific inhibitor of ubiquitin activating enzyme E1, inhibited ubiquitination of MC4R P272L and increased its cell surface expression and signaling to similar levels as wild-type MC4R. UBE41 had much less profound effects on MC4R I316S, another obesity-linked MC4R variant trapped in the ER. These data suggest that P272L is retained in the ER by a propensity to be ubiquitinated in the face of correct folding, which is only minimally shared by MC4R I316S. Thus, studies that combine clinical screening of obese patients and investigation of the functional defects of the obesity-linked MC4R variants can identify specific ways to correct these defects and are the first steps towards personalized medicine.

Exposure of MC4R to agonist in the endoplasmic reticulum stabilizes an active conformation of the receptor that does not desensitize

Significance Melanocortin-4 receptor (MC4R) is a cell-surface hormone receptor in the brain that is central to the control of appetite. Upstream pathways sensing energy balance in the organism lead to the secretion of α-melanocyte–stimulating hormone (α-MSH), which stimulates MC4R activity and leads to decreased appetite. Previously, it was found that MC4R becomes resistant to treatment with extracellular agonists by a mechanism that may involve desensitization of the receptor, arguing against their usefulness in treating obesity. In this study, we show that exposing MC4R to α-MSH in the endoplasmic reticulum leads to constant signaling of MC4R without desensitization. Our results indicate that the cellular localization where agonist binding initially takes place affects the conformation and the desensitization properties of MC4R, suggesting a target for therapy. Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in neurons of the hypothalamus where it regulates food intake. MC4R responds to an agonist, α-melanocyte–stimulating hormone (α-MSH) and to an antagonist/inverse agonist, agouti-related peptide (AgRP), which are released by upstream neurons. Binding to α-MSH leads to stimulation of receptor activity and suppression of food intake, whereas AgRP has opposite effects. MC4R cycles constantly between the plasma membrane and endosomes and undergoes agonist-mediated desensitization by being routed to lysosomes. MC4R desensitization and increased AgRP expression are thought to decrease the effectiveness of MC4R agonists as an antiobesity treatment. In this study, α-MSH, instead of being delivered extracellularly, is targeted to the endoplasmic reticulum (ER) of neuronal cells and cultured hypothalamic neurons. We find that the ER-targeted agonist associates with MC4R at this location, is transported to the cell surface, induces constant cAMP and AMP kinase signaling at maximal amplitude, abolishes desensitization of the receptor, and promotes both cell-surface expression and constant signaling by an obesity-linked MC4R variant, I316S, that otherwise is retained in the ER. Formation of the MC4R/agonist complex in the ER stabilizes the receptor in an active conformation that at the cell surface is insensitive to antagonism by AgRP and at the endosomes is refractory to routing to the lysosomes. The data indicate that targeting agonists to the ER can stabilize an active conformation of a G protein-coupled receptor that does not become desensitized, suggesting a target for therapy.

Inclusion bodies and autophagosomes: Are ER-derived protective organelles different than classical autophagosomes?

A hallmark of some endoplasmic reticulum (ER)-storage diseases is the formation of inclusion bodies (IBs) that are membrane-limited. The nature and function of the IBs has started to be investigated. We have recently found that sequestration of mutated α1-antitrypsin (ATZ) into IBs is a cell protective mechanism that maintains ER function. We also found that IBs are ER-derived and yet separate from the main ER and do not have markers of autophagosomes and lysosomes. We propose that formation of the IBs is a quality control mechanism that leads to storage of unwanted proteins outside the secretory pathway by a mechanism different than direct autophagosome formation from the ER. Addendum to: Granell S, Baldini G, Mohammad S, Nicolin V, Narducci P, Storrie B, Baldini G. Sequestration of mutated Éø-1-antitrypsin into inclusion bodies is a cell protective mechanism to maintain endoplasmic reticulum function. Mol Biol Cell 2008; 19:571-85.

Posttranslational regulation of tissue inhibitor of metalloproteinase-1 by calcium-dependent vesicular exocytosis.

Liver myofibroblasts derived from hepatic stellate cells (HSC) are critical mediators of liver fibrosis. Release of tissue inhibitor of metalloproteinase‐1 (TIMP‐1) advances liver fibrosis by blocking fibrinolysis. The mechanisms responsible for the posttranslational regulation of TIMP‐1 by myofibroblastic HSC are unknown. Here, we demonstrate that TIMP‐1 release by HSC is regulated in a posttranslational fashion via calcium‐sensitive vesicular exocytosis. To our knowledge, this is the first article to directly examine vesicular trafficking in myofibroblastic HSC, potentially providing a new target to treat and or prevent liver fibrosis.

Obesity-Linked Variants of Melanocortin-4 Receptor Are Misfolded in the Endoplasmic Reticulum and Can Be Rescued to the Cell Surface by a Chemical Chaperone

Melanocortin-4 receptor (MC4R) is a G protein-coupled receptor expressed in the brain where it controls food intake. Many obesity-linked MC4R variants are poorly expressed at the plasma membrane and are retained intracellularly. We have studied the intracellular localization of four obesity-linked MC4R variants, P78L, R165W, I316S, and I317T, in immortalized neurons. We find thatthesevariantsareallretainedintheendoplasmicreticulum(ER),areubiquitinatedtoagreaterextentthanthewild-type(wt) receptor,andinduceERstresswithincreasedlevelsofERchaperonesascomparedwithwt-MC4RandappearanceofCCAAT/enhancerbinding protein homologous protein. Expression of the X-box-binding-protein-1 with selective activation of a protective branch of the unfolded protein response did not have any effect on the cell surface expression of MC4R-I316S. Conversely, the pharmacological chaperone4-phenylbutyricacid(PBA)increasedthecellsurfaceexpressionofwt-MC4R,MC4R-I316S,andI317Tbymorethan40%.PBA decreased ubiquitination of MC4R-I316S and prevented ER stress induced by expression of the mutant, suggesting that the drug functionstopromoteMC4Rfolding.MC4R-I316Srescuedtothecellsurfaceisfunctional,witha52%increaseinagonist-inducedcAMP production, as compared with untreated cells. Also direct inhibition of wt-MC4R and MC4R-I316S ubiquitination by a specific inhibitor oftheubiquitin-activatingenzyme1increasedbyapproximately40%theexpressionofthereceptorsatthecellsurface,andtheeffects of PBA and ubiquitin-activating enzyme 1 were additive. These data offer a cell-based rationale that drugs that improve MC4R folding or decrease ER-associated degradation of the receptor may function to treat some forms of hereditary obesity.