RESUMO
Maintaining lipid composition diversity in membranes from different organelles is critical for numerous cellular processes. However, many lipids are synthesized in the endoplasmic reticulum (ER) and require delivery to other organelles. In this scenario, formation of membrane contact sites (MCS) between neighbouring organelles has emerged as a novel non-vesicular lipid transport mechanism. Dissecting the molecular composition of MCS identified phosphoinositides (PIs), cholesterol, scaffolding/tethering proteins as well as Ca2+ and Ca2+-binding proteins contributing to MCS functioning. Compelling evidence now exists for the shuttling of PIs and cholesterol across MCS, affecting their concentrations in distinct membrane domains and diverse roles in membrane trafficking. Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) at the plasma membrane (PM) not only controls endo-/exocytic membrane dynamics but is also critical in autophagy. Cholesterol is highly concentrated at the PM and enriched in recycling endosomes and Golgi membranes. MCS-mediated cholesterol transfer is intensely researched, identifying MCS dysfunction or altered MCS partnerships to correlate with de-regulated cellular cholesterol homeostasis and pathologies. Annexins, a conserved family of Ca2+-dependent phospholipid binding proteins, contribute to tethering and untethering events at MCS. In this chapter, we will discuss how Ca2+ homeostasis and annexins in the endocytic compartment affect the sensing and transfer of cholesterol and PIs across MCS.
Assuntos
Anexinas , Fosfatidilinositóis , Fosfatidilinositóis/metabolismo , Anexinas/metabolismo , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Transporte/metabolismo , Colesterol/metabolismoRESUMO
Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.
Assuntos
Anexina A6/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Hepatopatias/patologia , Longevidade , Animais , Comportamento Animal , Hepatopatias/etiologia , Hepatopatias/metabolismo , Camundongos , Camundongos Knockout , Proteína C1 de Niemann-PickRESUMO
The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.
Assuntos
Calmodulina/metabolismo , Membrana Celular/metabolismo , Animais , Células CHO , Sinalização do Cálcio/fisiologia , Linhagem Celular , Cricetulus , Ativação Enzimática/fisiologia , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Proteínas Tirosina Quinases/metabolismoRESUMO
BACKGROUND AND AIMS: Liver regeneration requires the organized and sequential activation of events that lead to restoration of hepatic mass. During this process, other vital liver functions need to be preserved, such as maintenance of blood glucose homeostasis, balancing the degradation of hepatic glycogen stores, and gluconeogenesis (GNG). Under metabolic stress, alanine is the main hepatic gluconeogenic substrate, and its availability is the rate-limiting step in this pathway. Na+ -coupled neutral amino acid transporters (SNATs) 2 and 4 are believed to facilitate hepatic alanine uptake. In previous studies, we demonstrated that a member of the Ca2+ -dependent phospholipid binding annexins, Annexin A6 (AnxA6), regulates membrane trafficking along endo- and exocytic pathways. Yet, although AnxA6 is abundantly expressed in the liver, its function in hepatic physiology remains unknown. In this study, we investigated the potential contribution of AnxA6 in liver regeneration. APPROACH AND RESULTS: Utilizing AnxA6 knockout mice (AnxA6-/- ), we challenged liver function after partial hepatectomy (PHx), inducing acute proliferative and metabolic stress. Biochemical and immunofluorescent approaches were used to dissect AnxA6-/- mice liver proliferation and energetic metabolism. Most strikingly, AnxA6-/- mice exhibited low survival after PHx. This was associated with an irreversible and progressive drop of blood glucose levels. Whereas exogenous glucose administration or restoration of hepatic AnxA6 expression rescued AnxA6-/- mice survival after PHx, the sustained hypoglycemia in partially hepatectomized AnxA6-/- mice was the consequence of an impaired alanine-dependent GNG in AnxA6-/- hepatocytes. Mechanistically, cytoplasmic SNAT4 failed to recycle to the sinusoidal plasma membrane of AnxA6-/- hepatocytes 48 hours after PHx, impairing alanine uptake and, consequently, glucose production. CONCLUSIONS: We conclude that the lack of AnxA6 compromises alanine-dependent GNG and liver regeneration in mice.
Assuntos
Anexina A6/metabolismo , Gluconeogênese/fisiologia , Regeneração Hepática/fisiologia , Animais , Anexina A6/genética , Membrana Celular/metabolismo , Modelos Animais de Doenças , Glucose/metabolismo , Glicólise/fisiologia , Hepatectomia , Hepatócitos/metabolismo , Humanos , Fígado/citologia , Fígado/metabolismo , Fígado/cirurgia , Masculino , Camundongos , Camundongos KnockoutRESUMO
Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.
Assuntos
Anexina A6/genética , Colesterol/genética , Proteínas Ativadoras de GTPase/genética , Doença de Niemann-Pick Tipo C/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Células CHO , Proteínas de Transporte/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Proteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Domínios Proteicos/genética , Transporte Proteico/genética , RNA Interferente Pequeno/genética , proteínas de unión al GTP Rab7RESUMO
Calmodulin is a ubiquitous signalling protein that controls many biological processes due to its capacity to interact and/or regulate a large number of cellular proteins and pathways, mostly in a Ca2+-dependent manner. This complex interactome of calmodulin can have pleiotropic molecular consequences, which over the years has made it often difficult to clearly define the contribution of calmodulin in the signal output of specific pathways and overall biological response. Most relevant for this review, the ability of calmodulin to influence the spatiotemporal signalling of several small GTPases, in particular KRas and Rac1, can modulate fundamental biological outcomes such as proliferation and migration. First, direct interaction of calmodulin with these GTPases can alter their subcellular localization and activation state, induce post-translational modifications as well as their ability to interact with effectors. Second, through interaction with a set of calmodulin binding proteins (CaMBPs), calmodulin can control the capacity of several guanine nucleotide exchange factors (GEFs) to promote the switch of inactive KRas and Rac1 to an active conformation. Moreover, Rac1 is also an effector of KRas and both proteins are interconnected as highlighted by the requirement for Rac1 activation in KRas-driven tumourigenesis. In this review, we attempt to summarize the multiple layers how calmodulin can regulate KRas and Rac1 GTPases in a variety of cellular events, with biological consequences and potential for therapeutic opportunities in disease settings, such as cancer.
Assuntos
Calmodulina/metabolismo , Carcinogênese/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Calmodulina/genética , Carcinogênese/genética , Pleiotropia Genética , Humanos , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genéticaRESUMO
The endocytic compartment is not only the functional continuity of the plasma membrane but consists of a diverse collection of intracellular heterogeneous complex structures that transport, amplify, sustain, and/or sort signaling molecules. Over the years, it has become evident that early, late, and recycling endosomes represent an interconnected vesicular-tubular network able to form signaling platforms that dynamically and efficiently translate extracellular signals into biological outcome. Cell activation, differentiation, migration, death, and survival are some of the endpoints of endosomal signaling. Hence, to understand the role of the endosomal system in signal transduction in space and time, it is therefore necessary to dissect and identify the plethora of decoders that are operational in the different steps along the endocytic pathway. In this chapter, we focus on the regulation of spatiotemporal signaling in cells, considering endosomes as central platforms, in which several small GTPases proteins of the Ras superfamily, in particular Ras and Rac1, actively participate to control cellular processes like proliferation and cell mobility.
Assuntos
Proliferação de Células/genética , Endossomos/genética , Proteínas rac1 de Ligação ao GTP/genética , Proteínas ras/genética , Movimento Celular/genética , Endocitose/genética , Humanos , Transporte Proteico , Transdução de Sinais/genéticaRESUMO
Annexins are a family of proteins that bind to phospholipids in a calcium-dependent manner. Earlier studies implicated annexin A6 (AnxA6) to inhibit secretion and participate in the organization of the extracellular matrix. We recently showed that elevated AnxA6 levels significantly reduced secretion of the extracellular matrix protein fibronectin (FN). Because FN is directly linked to the ability of cells to migrate, this prompted us to investigate the role of AnxA6 in cell migration. Up-regulation of AnxA6 in several cell models was associated with reduced cell migration in wound healing, individual cell tracking and three-dimensional migration/invasion assays. The reduced ability of AnxA6-expressing cells to migrate was associated with decreased cell surface expression of αVß3 and α5ß1 integrins, both FN receptors. Mechanistically, we found that elevated AnxA6 levels interfered with syntaxin-6 (Stx6)-dependent recycling of integrins to the cell surface. AnxA6 overexpression caused mislocalization and accumulation of Stx6 and integrins in recycling endosomes, whereas siRNA-mediated AnxA6 knockdown did not modify the trafficking of integrins. Given our recent findings that inhibition of cholesterol export from late endosomes (LEs) inhibits Stx6-dependent integrin recycling and that elevated AnxA6 levels cause LE cholesterol accumulation, we propose that AnxA6 and blockage of LE cholesterol transport are critical for endosomal function required for Stx6-mediated recycling of integrins in cell migration.
Assuntos
Anexina A6/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/metabolismo , Proteínas Qa-SNARE/metabolismo , Animais , Anexina A6/antagonistas & inibidores , Anexina A6/genética , Células CHO , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Movimento Celular , Células Cultivadas , Cricetulus , Endossomos/ultraestrutura , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfaVbeta3/antagonistas & inibidores , Camundongos , Microscopia Confocal , Microscopia de Vídeo , Proteínas Qa-SNARE/antagonistas & inibidores , Proteínas Qa-SNARE/genética , Interferência de RNA , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Imagem com Lapso de TempoRESUMO
Niemann-Pick C disease is a neurovisceral disorder caused by mutations in the NPC gene that result in systemic accumulation of intracellular cholesterol. Although neurodegeneration defines the disease's severity, in most patients it is preceded by hepatic complications such as cholestatic jaundice or hepatomegaly. To analyze the contribution of the hepatic disease in Niemann-Pick C disease progression and to evaluate the degree of primary and secondary hepatic damage, we generated a transgenic mouse with liver-selective expression of NPC1 from embryonic stages. Hepatic NPC1 re-expression did not ameliorate the onset and progression of neurodegeneration of the NPC1-null animal. However, the mice showed reduced hepatomegalia and dramatic, although not complete, reduction of hepatic cholesterol and serum bile salts, bilirubin, and transaminase levels. Therefore, hepatic primary and secondary cholesterol deposition and damage occur simultaneously during Niemann-Pick C disease progression.
Assuntos
Colesterol/metabolismo , Modelos Animais de Doenças , Hepatopatias/complicações , Fígado/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Proteínas/genética , Animais , Ácidos e Sais Biliares/sangue , Bilirrubina/sangue , Colesterol/análise , Progressão da Doença , Células-Tronco Embrionárias , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Fígado/patologia , Hepatopatias/genética , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Knockout , Proteína C1 de Niemann-Pick , Doença de Niemann-Pick Tipo C/complicações , Doença de Niemann-Pick Tipo C/genética , Doença de Niemann-Pick Tipo C/patologia , Proteínas/metabolismo , Transaminases/sangueRESUMO
Despite the discovery of annexins 40 years ago, we are just beginning to understand some of the functions of these still enigmatic proteins. Defined and characterized by their ability to bind anionic membrane lipids in a Ca2+-dependent manner, each annexin has to be considered a multifunctional protein, with a multitude of cellular locations and diverse activities. Underlying causes for this considerable functional diversity include their capability to associate with multiple cytosolic and membrane proteins. In recent years, the increasingly recognized establishment of membrane contact sites between subcellular compartments opens a new scenario for annexins as instrumental players to link Ca2+ signalling with the integration of membrane trafficking in many facets of cell physiology. In this chapter, we review and discuss current knowledge on the contribution of annexins in the biogenesis and functioning of the late endocytic compartment, affecting endo- and exocytic pathways in a variety of physiological consequences ranging from membrane repair, lysosomal exocytosis, to cell migration.
Assuntos
Anexinas/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Animais , Movimento Celular/fisiologia , Endocitose/fisiologia , Exocitose/fisiologia , Humanos , Lisossomos/metabolismo , Lipídeos de Membrana/metabolismoRESUMO
Activating mutations in the K-Ras small GTPase are extensively found in human tumors. Although these mutations induce the generation of a constitutively GTP-loaded, active form of K-Ras, phosphorylation at Ser181 within the C-terminal hypervariable region can modulate oncogenic K-Ras function without affecting the in vitro affinity for its effector Raf-1. In striking contrast, K-Ras phosphorylated at Ser181 shows increased interaction in cells with the active form of Raf-1 and with p110α, the catalytic subunit of PI 3-kinase. Because the majority of phosphorylated K-Ras is located at the plasma membrane, different localization within this membrane according to the phosphorylation status was explored. Density-gradient fractionation of the plasma membrane in the absence of detergents showed segregation of K-Ras mutants that carry a phosphomimetic or unphosphorylatable serine residue (S181D or S181A, respectively). Moreover, statistical analysis of immunoelectron microscopy showed that both phosphorylation mutants form distinct nanoclusters that do not overlap. Finally, induction of oncogenic K-Ras phosphorylation - by activation of protein kinase C (PKC) - increased its co-clustering with the phosphomimetic K-Ras mutant, whereas (when PKC is inhibited) non-phosphorylated oncogenic K-Ras clusters with the non-phosphorylatable K-Ras mutant. Most interestingly, PI 3-kinase (p110α) was found in phosphorylated K-Ras nanoclusters but not in non-phosphorylated K-Ras nanoclusters. In conclusion, our data provide - for the first time - evidence that PKC-dependent phosphorylation of oncogenic K-Ras induced its segregation in spatially distinct nanoclusters at the plasma membrane that, in turn, favor activation of Raf-1 and PI 3-kinase.
Assuntos
Genes ras , Proteínas ras/genética , Proteínas ras/metabolismo , Membrana Celular/genética , Membrana Celular/metabolismo , Células HEK293 , Células HeLa , Humanos , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-raf/metabolismo , Transdução de SinaisRESUMO
The endocytic compartment is emerging as a functional platform for controlling important cellular processes. We have found that â¼10 to 15% of total KRas, a protein that is frequently mutated in cancer, is present on endosomes, independent of its activation state. The dynamics of GFP-KRas wild-type (WT) and constitutively active or inactive mutants on endosomes were analyzed by fluorescence recovery after photobleaching (FRAP) microscopy. The measurements revealed an extraordinarily fast recovery of KRas WT [half-time (HT), â¼1.3 s] compared to HRas, Rab5, and EGFR, with the active KRasG12V mutant being significantly faster and more mobile (HT, â¼1 s, and â¼82% of exchangeable fraction) than the inactive KRasS17N (HT, â¼1.6 s, and â¼60% of exchangeable fraction). KRas rapidly switches from the cytoplasm to the endosomal membranes by an electrostatic interaction between its polybasic region and the endosomal acidic phospholipids, mainly phosphatidylserine.-Gelabert-Baldrich, M., Soriano-Castell, D., Calvo, M., Lu, A., Viña-Vilaseca, A., Rentero, C., Pol, A., Grinstein, S. Enrich, C., Tebar, F. Dynamics of KRas on endosomes: involvement of acidic phospholipids in its association.
Assuntos
Endossomos/metabolismo , Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Células COS , Células Cultivadas , Chlorocebus aethiops , Citoplasma/genética , Citoplasma/metabolismo , Endocitose/genética , Endocitose/fisiologia , Endossomos/genética , Fibroblastos/metabolismo , Recuperação de Fluorescência Após Fotodegradação , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Antimicrobial peptides are an interesting source of non-cytotoxic drug delivery vectors. Herein, we report on the identification of a new cell-penetrating peptide (KKLFKKILKKL-NH2, BP16) from a set of antimicrobial peptides selected from a library of cecropin-melittin hybrids (CECMEL11) previously designed to be used in plant protection. This set of peptides was screened for their cytotoxicity against breast adenocarcinoma MCF-7, pancreas adenocarcinoma CAPAN-1 and mouse embryonic fibroblast 3T3 cell lines. BP16 resulted to be non-toxic against both malignant and non-malignant cells at concentrations up to 200 µM. We demonstrated by flow cytometry and confocal microscopy that BP16 is mainly internalized in the cells through a clathrin dependent endocytosis and that it efficiently accumulates in the cell cytoplasm. We confirmed that the cell-penetrating properties of BP16 are retained after conjugating it to the breast tumor homing peptide CREKA. Furthermore, we assessed the potential of BP16 as a drug delivery vector by conjugating the anticancer drug chlorambucil to BP16 and to a CREKA-BP16 conjugate. The efficacy of the drug increased between 6 and 9 times when conjugated to BP16 and between 2 and 4.5 times when attached to the CREKA-BP16 derivative. The low toxicity and the excellent cell-penetrating properties clearly suggest that BP16 is a suitable vector for the delivery of therapeutic agents into cells.
Assuntos
Antineoplásicos/farmacologia , Peptídeos Penetradores de Células/farmacologia , Sistemas de Liberação de Medicamentos , Células 3T3 , Animais , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Peptídeos Penetradores de Células/síntese química , Peptídeos Penetradores de Células/química , Relação Dose-Resposta a Droga , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Células MCF-7 , Camundongos , Relação Estrutura-AtividadeRESUMO
Ir(III) and Ru(II) polypyridyl complexes are promising photosensitizers (PSs) for photodynamic therapy (PDT) due to their outstanding photophysical properties. Herein, one series of cyclometallated Ir(III) complexes and two series of Ru(II) polypyridyl derivatives bearing three different thiazolyl-ß-carboline N^N' ligands have been synthesized, aiming to evaluate the impact of the different metal fragments ([Ir(C^N)2]+ or [Ru(N^N)2]2+) and N^N' ligands on the photophysical and biological properties. All the compounds exhibit remarkable photostability under blue-light irradiation and are emissive (605 < λem < 720 nm), with the Ru(II) derivatives displaying higher photoluminescence quantum yields and longer excited state lifetimes. The Ir PSs display pKa values between 5.9 and 7.9, whereas their Ru counterparts are less acidic (pKa > 9.3). The presence of the deprotonated form in the Ir-PSs favours the generation of reactive oxygen species (ROS) since, according to theoretical calculations, it features a low-lying ligand-centered triplet excited state (T1 = 3LC) with a long lifetime. All compounds have demonstrated anticancer activity. Ir(III) complexes 1-3 exhibit the highest cytotoxicity in dark conditions, comparable to cisplatin. Their activity is notably enhanced by blue-light irradiation, resulting in nanomolar IC50 values and phototoxicity indexes (PIs) between 70 and 201 in different cancer cell lines. The Ir(III) PSs are also activated by green (with PI between 16 and 19.2) and red light in the case of complex 3 (PI = 8.5). Their antitumor efficacy is confirmed by clonogenic assays and using spheroid models. The Ir(III) complexes rapidly enter cells, accumulating in mitochondria and lysosomes. Upon photoactivation, they generate ROS, leading to mitochondrial dysfunction and lysosomal damage and ultimately cell apoptosis. Additionally, they inhibit cancer cell migration, a crucial step in metastasis. In contrast, Ru(II) complex 6 exhibits moderate mitochondrial activity. Overall, Ir(III) complexes 1-3 show potential for selective light-controlled cancer treatment, providing an alternative mechanism to chemotherapy and the ability to inhibit lethal cancer cell dissemination.
Assuntos
Antineoplásicos , Carbolinas , Complexos de Coordenação , Ensaios de Seleção de Medicamentos Antitumorais , Irídio , Fotoquimioterapia , Fármacos Fotossensibilizantes , Rutênio , Humanos , Irídio/química , Irídio/farmacologia , Rutênio/química , Rutênio/farmacologia , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/síntese química , Ligantes , Fármacos Fotossensibilizantes/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/síntese química , Complexos de Coordenação/farmacologia , Complexos de Coordenação/química , Complexos de Coordenação/síntese química , Carbolinas/química , Carbolinas/farmacologia , Carbolinas/síntese química , Estrutura Molecular , Relação Estrutura-Atividade , Proliferação de Células/efeitos dos fármacos , Piridinas/química , Piridinas/farmacologia , Piridinas/síntese química , Espécies Reativas de Oxigênio/metabolismo , Relação Dose-Resposta a Droga , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Apoptose/efeitos dos fármacosRESUMO
The main cellular Ca(2+) sensor, calmodulin (CaM), interacts with and regulates several small GTPases, including Rac1. The present study revealed high binding affinity of Rac1 for CaM and uncovered two new essential binding domains in Rac1: the polybasic region, important for phosphatidylinositol-4-phosphate 5-kinase (PIP5K) interaction, and the adjacent prenyl group. CaM inhibition increased Rac1 binding to PIP5K and induced an extensive phosphatidylinositol 4,5-bisphosphate (PI4,5P(2) )-positive tubular membrane network. Immunofluorescence demonstrated that the tubules were plasma membrane invaginations resulting from an ADP-ribosylation factor 6 (ARF6)-dependent and clathrin-independent pathway. The role of Rac1 in this endocytic route was analyzed by expressing constitutively active and inactive mutants. While active Rac1 impaired tubulation, the inactive mutant enhanced it. Intriguingly, inactive mutant expression elicited tubulation by recruiting PIP5K and inhibiting Rac1 at the plasma membrane. Accordingly, CaM inhibition inactivated Rac1 and increased Rac1/PIP5K interaction. Therefore, our findings highlight an important new role for Rac1 and CaM in controlling clathrin-independent endocytosis.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Calmodulina/metabolismo , Endocitose/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Fator 6 de Ribosilação do ADP , Animais , Células COS , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Clatrina/metabolismo , Camundongos , Células NIH 3T3 , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Células VeroRESUMO
Caveolin-1 (CAV1) and CAV3 are membrane-sculpting proteins driving the formation of the plasma membrane (PM) caveolae. Within the PM mosaic environment, caveola assembly is unique as it requires progressive oligomerization of newly synthesized caveolins while trafficking through the biosynthetic-secretory pathway. Here, we have investigated these early events by combining structural, biochemical, and microscopy studies. We uncover striking trafficking differences between caveolins, with CAV1 rapidly exported to the Golgi and PM while CAV3 is initially retained in the endoplasmic reticulum and laterally moves into lipid droplets. The levels of caveolins in the endoplasmic reticulum are controlled by proteasomal degradation, and only monomeric/low oligomeric caveolins are exported into the cis-Golgi with higher-order oligomers assembling beyond this compartment. When any of those early proteostatic mechanisms are compromised, chemically or genetically, caveolins tend to accumulate along the secretory pathway forming non-functional aggregates, causing organelle damage and triggering cellular stress. Accordingly, we propose a model in which disrupted proteostasis of newly synthesized caveolins contributes to pathogenesis.
Assuntos
Caveolinas , Proteostase , Caveolinas/metabolismo , Caveolina 1/metabolismo , Proteínas de Membrana/metabolismo , Cavéolas/metabolismo , Membrana Celular/metabolismo , Complexo de Golgi/metabolismoRESUMO
As cells migrate and experience forces from their surroundings, they constantly undergo mechanical deformations which reshape their plasma membrane (PM). To maintain homeostasis, cells need to detect and restore such changes, not only in terms of overall PM area and tension as previously described, but also in terms of local, nanoscale topography. Here, we describe a novel phenomenon, by which cells sense and restore mechanically induced PM nanoscale deformations. We show that cell stretch and subsequent compression reshape the PM in a way that generates local membrane evaginations in the 100 nm scale. These evaginations are recognized by I-BAR proteins, which triggers a burst of actin polymerization mediated by Rac1 and Arp2/3. The actin polymerization burst subsequently re-flattens the evagination, completing the mechanochemical feedback loop. Our results demonstrate a new mechanosensing mechanism for PM shape homeostasis, with potential applicability in different physiological scenarios.
Assuntos
Actinas , Actinas/metabolismo , Membrana Celular/metabolismo , HomeostaseRESUMO
Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.
Assuntos
Anexina A6/metabolismo , LDL-Colesterol/metabolismo , Adesões Focais/metabolismo , Proteína C1 de Niemann-Pick/metabolismo , proteínas de unión al GTP Rab7/metabolismo , Animais , Células CHO , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Cricetulus , Humanos , Proteínas de Membrana/metabolismoRESUMO
In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes.
Assuntos
Lipídeos/química , Proteínas/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Dados de Sequência MolecularRESUMO
Membrane contact sites (MCS) are specialized small areas of close apposition between two different organelles that have led researchers to reconsider the dogma of intercellular communication via vesicular trafficking. The latter is now being challenged by the discovery of lipid and ion transfer across MCS connecting adjacent organelles. These findings gave rise to a new concept that implicates cell compartments not to function as individual and isolated entities, but as a dynamic and regulated ensemble facilitating the trafficking of lipids, including cholesterol, and ions. Hence, MCS are now envisaged as metabolic platforms, crucial for cellular homeostasis. In this context, well-known as well as novel proteins were ascribed functions such as tethers, transporters, and scaffolds in MCS, or transient MCS companions with yet unknown functions. Intriguingly, we and others uncovered metabolic alterations in cell-based disease models that perturbed MCS size and numbers between coupled organelles such as endolysosomes, the endoplasmic reticulum, mitochondria, or lipid droplets. On the other hand, overexpression or deficiency of certain proteins in this narrow 10-30 nm membrane contact zone can enable MCS formation to either rescue compromised MCS function, or in certain disease settings trigger undesired metabolite transport. In this "Mini Review" we summarize recent findings regarding a subset of annexins and discuss their multiple roles to regulate MCS dynamics and functioning. Their contribution to novel pathways related to MCS biology will provide new insights relevant for a number of human diseases and offer opportunities to design innovative treatments in the future.