RESUMO
The toll-like receptor 4 (TLR4) is a central regulator of innate immunity that primarily recognizes bacterial lipopolysaccharide cell wall constituents to trigger cytokine secretion. We identify the intramembrane protease RHBDL4 as a negative regulator of TLR4 signaling. We show that RHBDL4 triggers degradation of TLR4's trafficking factor TMED7. This counteracts TLR4 transport to the cell surface. Notably, TLR4 activation mediates transcriptional upregulation of RHBDL4 thereby inducing a negative feedback loop to reduce TLR4 trafficking to the plasma membrane. This secretory cargo tuning mechanism prevents the over-activation of TLR4-dependent signaling in an in vitro Mycobacterium tuberculosis macrophage infection model and consequently alleviates septic shock in a mouse model. A hypomorphic RHBDL4 mutation linked to Kawasaki syndrome, an ill-defined inflammatory disorder in children, further supports the pathophysiological relevance of our findings. In this work, we identify an RHBDL4-mediated axis that acts as a rheostat to prevent over-activation of the TLR4 pathway.
Assuntos
Transdução de Sinais , Receptor 4 Toll-Like , Animais , Criança , Humanos , Camundongos , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Regulação para Baixo , Lipopolissacarídeos/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Intramembrane proteolysis regulates important processes such as signaling and transcriptional and posttranslational abundance control of proteins with key functions in metabolic pathways. This includes transcriptional control of mevalonate pathway genes, thereby ensuring balanced biosynthesis of cholesterol and other isoprenoids. Our work shows that, at high cholesterol levels, signal peptide peptidase (SPP) cleaves squalene synthase (SQS), an enzyme that defines the branching point for allocation of isoprenoids to the sterol and nonsterol arms of the mevalonate pathway. This intramembrane cleavage releases SQS from the membrane and targets it for proteasomal degradation. Regulation of this mechanism is achieved by the E3 ubiquitin ligase TRC8 that, in addition to ubiquitinating SQS in response to cholesterol levels, acts as an allosteric activator of SPP-catalyzed intramembrane cleavage of SQS. Cellular cholesterol levels increase in the absence of SPP activity. We infer from these results that, SPP-TRC8 mediated abundance control of SQS acts as a regulation step within the mevalonate pathway.
Assuntos
Farnesil-Difosfato Farnesiltransferase , Ácido Mevalônico , Ácido Aspártico Endopeptidases , Colesterol/metabolismo , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácido Mevalônico/metabolismo , Terpenos , Células HEK293 , HumanosRESUMO
The mitochondrial rhomboid protease PARL regulates mitophagy by balancing intramembrane proteolysis of PINK1 and PGAM5. It has been implicated in the pathogenesis of Parkinson's disease, but its investigation as a possible therapeutic target is challenging in this context because genetic deficiency of PARL may result in compensatory mechanisms. To address this problem, we undertook a hitherto unavailable chemical biology strategy. We developed potent PARL-targeting ketoamide inhibitors and investigated the effects of acute PARL suppression on the processing status of PINK1 intermediates and on Parkin activation. This approach revealed that PARL inhibition leads to a robust activation of the PINK1/Parkin pathway without major secondary effects on mitochondrial properties, which demonstrates that the pharmacological blockage of PARL to boost PINK1/Parkin-dependent mitophagy is a feasible approach to examine novel therapeutic strategies for Parkinson's disease. More generally, this study showcases the power of ketoamide inhibitors for cell biological studies of rhomboid proteases.
Assuntos
Doença de Parkinson , Peptídeo Hidrolases , Humanos , Metaloproteases/genética , Metaloproteases/metabolismo , Mitofagia , Doença de Parkinson/tratamento farmacológico , Proteínas Quinases/metabolismo , Proteínas Mitocondriais/metabolismo , Endopeptidases , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Cells need to detect and degrade faulty membrane proteins to maintain homeostasis. In this study, we identify a previously unknown function of the human signal peptidase complex (SPC)-the enzyme that removes endoplasmic reticulum (ER) signal peptides-as a membrane protein quality control factor. We show that the SPC cleaves membrane proteins that fail to correctly fold or assemble into their native complexes at otherwise hidden cleavage sites, which our study reveals to be abundant in the human membrane proteome. This posttranslocational cleavage synergizes with ER-associated degradation to sustain membrane protein homeostasis and contributes to cellular fitness. Cryptic SPC cleavage sites thus serve as predetermined breaking points that, when exposed, help to target misfolded or surplus proteins for degradation, thereby maintaining a healthy membrane proteome.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Proteínas de Membrana , Serina Endopeptidases , Humanos , Proteínas de Membrana/metabolismo , Proteoma , ProteóliseRESUMO
Membrane thinning by rhomboid proteins has been proposed to reduce hydrophobic mismatch, providing a unique environment for important functions ranging from intramembrane proteolysis to retrotranslocation in protein degradation. We show by in vitro reconstitution and solid-state nuclear magnetic resonance that the lipid environment of the Escherichia coli rhomboid protease GlpG influences its activity with an optimal hydrophobic membrane thickness between 24 and 26 Å. While phosphatidylcholine membranes are only negligibly altered by GlpG, in an E. coli-relevant lipid mix of phosphatidylethanolamine and phosphatidylglycerol, a thinning by 1.1 Å per leaflet is observed. Protease activity is strongly correlated with membrane thickness and shows no lipid headgroup specificity. We infer from these results that, by adjusting the thickness of specific membrane domains, membrane proteins shape the bilayer for their specific needs.
RESUMO
Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.
Assuntos
Complexo de Golgi , Doenças por Armazenamento dos Lisossomos , Lisossomos , Proteínas , Animais , Complexo de Golgi/metabolismo , Humanos , Doenças por Armazenamento dos Lisossomos/genética , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Camundongos , Transporte Proteico , Proteínas/genética , Proteínas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Protein degradation is fundamentally important to ensure cell homeostasis. In the endoplasmic reticulum (ER), the ER-associated degradation (ERAD) pathway targets incorrectly folded and unassembled proteins for turnover by the cytoplasmic proteasome. Previously, we showed that the rhomboid protease RHBDL4, together with p97, mediates membrane protein degradation. However, whether RHBDL4 acts in concert with additional ERAD components is unclear, and its full substrate spectrum remains to be defined. Here, we show that, in addition to membrane proteins, RHBDL4 cleaves aggregation-prone luminal ERAD substrates. Since mutations of the RHBDL4 rhomboid domain led to stabilization of substrates at the cytoplasmic side, we hypothesize that, analogous to the homolog ERAD factor derlin, RHBDL4 is directly involved in substrate retrotranslocation. RHBDL4's interaction with the erlin ERAD complex and reciprocal interaction of rhomboid substrates with erlins suggest that RHBDL4 and erlins form a complex that clips substrates and thereby rescues aggregation-prone peptides in the ER from aggregation.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Retículo Endoplasmático , Endopeptidases/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , ProteóliseRESUMO
The intramembrane protease PARL acts as a crucial mitochondrial safeguard by cleaving the mitophagy regulators PINK1 and PGAM5. Depending on the stress level, PGAM5 can either stimulate cell survival or cell death. In contrast to PINK1, which is constantly cleaved in healthy mitochondria and only active when the inner mitochondrial membrane is depolarized, PGAM5 processing is inversely regulated. However, determinants of PGAM5 that indicate it as a conditional substrate for PARL have not been rigorously investigated, and it is unclear how uncoupling the mitochondrial membrane potential affects its processing compared to that of PINK1. Here, we show that several polar transmembrane residues in PGAM5 distant from the cleavage site serve as determinants for its PARL-catalyzed cleavage. Our NMR analysis indicates that a short N-terminal amphipathic helix, followed by a kink and a C-terminal transmembrane helix harboring the scissile peptide bond are key for a productive interaction with PARL. Furthermore, we also show that PGAM5 is stably inserted into the inner mitochondrial membrane until uncoupling the membrane potential triggers its disassembly into monomers, which are then cleaved by PARL. In conclusion, we propose a model in which PGAM5 is slowly processed by PARL-catalyzed cleavage that is influenced by multiple hierarchical substrate features, including a membrane potential-dependent oligomeric switch.
Assuntos
Homeostase , Metaloproteases , Mitocôndrias , Proteínas Mitocondriais , Fosfoproteínas Fosfatases , Proteólise , Células HeLa , Humanos , Metaloproteases/metabolismo , Mitocôndrias/enzimologia , Proteínas Mitocondriais/metabolismo , Peptídeos/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Proteínas Quinases/metabolismoRESUMO
Precise distribution of proteins is essential to sustain the viability of cells. A complex network of protein synthesis and targeting factors cooperate with protein quality control systems to ensure protein homeostasis. Defective proteins are inevitably degraded by the ubiquitin-proteasome system and lysosomes. However, due to overlapping targeting information and limited targeting fidelity, certain proteins become mislocalized. In this review, we present the idea that transmembrane dislocases recognize and remove mislocalized membrane proteins from cellular organelles. This enables other targeting attempts and prevents degradation of mislocalized but otherwise functional proteins. These transmembrane dislocases can be found in the outer mitochondrial membrane (OMM) and endoplasmic reticulum (ER). We highlight common principles regarding client recognition and outline open questions in our understanding of transmembrane dislocases.
Assuntos
Retículo Endoplasmático , Membranas Mitocondriais , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Membranas Mitocondriais/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte ProteicoRESUMO
Protein homeostasis mechanisms are fundamentally important to match cellular needs and to counteract stress conditions. A fundamental challenge is to understand how defective proteins are recognized and extracted from cellular organelles to be degraded in the cytoplasm. The endoplasmic reticulum (ER)-associated degradation (ERAD) pathway is the best-understood organellar protein quality control system. Here, we review new insights into the mechanism of recognition and retrotranslocation of client proteins in ERAD. In addition to the membrane-integral ERAD E3 ubiquitin ligases, we highlight one protein family that is remarkably often involved in various aspects of membrane protein quality control and protein dislocation: the rhomboid superfamily, which includes derlins and intramembrane serine proteases. Rhomboid-like proteins have been found to control protein homeostasis in the ER, but also in other eukaryotic organelles and in bacteria, pointing toward conserved principles of membrane protein quality control across organelles and evolution.
Assuntos
Degradação Associada com o Retículo Endoplasmático/fisiologia , Proteostase/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Células Eucarióticas/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The rhomboid protease PARL is a critical regulator of mitochondrial homeostasis through its cleavage of substrates such as PINK1, PGAM5, and Smac/Diablo, which have crucial roles in mitochondrial quality control and apoptosis. However, the catalytic properties of PARL, including the effect of lipids on the protease, have never been characterized in vitro. To address this, we isolated human PARL expressed in yeast and used FRET-based kinetic assays to measure proteolytic activity in vitro. We show that PARL activity in detergent is enhanced by cardiolipin, a lipid enriched in the mitochondrial inner membrane. Significantly higher turnover rates were observed for PARL reconstituted in proteoliposomes, with Smac/Diablo being cleaved most rapidly at a rate of 1 min-1. In contrast, PGAM5 is cleaved with the highest efficiency (kcat/KM) compared with PINK1 and Smac/Diablo. In proteoliposomes, a truncated ß-cleavage form of PARL, a physiological form known to affect mitochondrial fragmentation, is more active than the full-length enzyme for hydrolysis of PINK1, PGAM5, and Smac/Diablo. Multiplex profiling of 228 peptides reveals that PARL prefers substrates with a bulky side chain such as Phe in P1, which is distinct from the preference for small side chain residues typically found with bacterial rhomboid proteases. This study using recombinant PARL provides fundamental insights into its catalytic activity and substrate preferences that enhance our understanding of its role in mitochondrial function and has implications for specific inhibitor design.
Assuntos
Metaloproteases/metabolismo , Metaloproteases/fisiologia , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/fisiologia , Proteínas Reguladoras de Apoptose/metabolismo , Domínio Catalítico , Endopeptidases/metabolismo , Células HEK293 , Células HeLa , Humanos , Metaloproteases/genética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Peptídeo Hidrolases/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , ProteóliseRESUMO
Interleukin-11 (IL-11) is a pleiotropic cytokine with both pro- and anti-inflammatory properties. It activates its target cells via binding to the membrane-bound IL-11 receptor (IL-11R), which then recruits a homodimer of the ubiquitously expressed, signal-transducing receptor gp130. Besides this classic signaling pathway, IL-11 can also bind to soluble forms of the IL-11R (sIL-11R), and IL-11/sIL-11R complexes activate cells via the induction of gp130 homodimerization (trans-signaling). We have previously reported that the metalloprotease ADAM10 cleaves the membrane-bound IL-11R and thereby generates sIL-11R. In this study, we identify the rhomboid intramembrane protease RHBDL2 as a so far unrecognized alternative sheddase that can efficiently trigger IL-11R secretion. We determine the cleavage site used by RHBDL2, which is located in the extracellular part of the receptor in close proximity to the plasma membrane, between Ala-370 and Ser-371. Furthermore, we identify critical amino acid residues within the transmembrane helix that are required for IL-11R proteolysis. We also show that ectopically expressed RHBDL2 is able to cleave the IL-11R within the early secretory pathway and not only at the plasma membrane, indicating that its subcellular localization plays a central role in controlling its activity. Moreover, RHBDL2-derived sIL-11R is biologically active and able to perform IL-11 trans-signaling. Finally, we show that the human mutation IL-11R-A370V does not impede IL-11 classic signaling, but prevents RHBDL2-mediated IL-11R cleavage.
Assuntos
Interleucina-11/fisiologia , Receptores de Interleucina-11/metabolismo , Serina Endopeptidases/fisiologia , Células HEK293 , Células HeLa , Humanos , Proteólise , Receptores de Interleucina-11/química , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Iron export via the transport protein ferroportin (Fpn) plays a critical role in the regulation of dietary iron absorption and iron recycling in macrophages. Fpn plasma membrane expression is controlled by the hepatic iron-regulated hormone hepcidin in response to high iron availability and inflammation. Hepcidin binds to the central cavity of the Fpn transporter to block iron export either directly or by inducing Fpn internalization and lysosomal degradation. Here, we investigated whether iron deficiency affects Fpn protein turnover. METHODS: We ectopically expressed Fpn in HeLa cells and used cycloheximide chase experiments to study basal and hepcidin-induced Fpn degradation under extracellular and intracellular iron deficiency. CONCLUSIONS/GENERAL SIGNIFICANCE: We show that iron deficiency does not affect basal Fpn turnover but causes a significant delay in hepcidin-induced degradation when cytosolic iron levels are low. These data have important mechanistic implications supporting the hypothesis that iron export is required for efficient targeting of Fpn by hepcidin. Additionally, we show that Fpn degradation is not involved in protecting cells from intracellular iron deficiency.
Assuntos
Proteínas de Transporte de Cátions/genética , Hepcidinas/genética , Deficiências de Ferro , Proteínas de Transporte de Cátions/metabolismo , Cicloeximida/farmacologia , Desferroxamina/farmacologia , Regulação da Expressão Gênica , Células HeLa , Hepcidinas/metabolismo , Humanos , Transporte de Íons/efeitos dos fármacos , Quelantes de Ferro/farmacologia , Ligação Proteica/efeitos dos fármacos , Inibidores da Síntese de Proteínas/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Transdução de SinaisRESUMO
Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Marcação de Genes/métodos , Reação em Cadeia da Polimerase/métodos , Alelos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular , Células Cultivadas , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Genes Reporter , Sequenciamento de Nucleotídeos em Larga Escala , Recombinação Homóloga , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Oligonucleotídeos/genética , RNA Guia de Cinetoplastídeos/genética , TransfecçãoRESUMO
Intramembrane proteases catalyze the unusual cleavage of peptide bonds in the plane of biological membranes. They are categorized according to their active site. The GxGD aspartyl proteases comprise presenilin, the signal peptide peptidase (SPP), and SPP-like (SPPL) proteases. Here we focus on the functionally related SPP and SPPL proteases, and review the current understanding of their substrate specificity and summarize known physiological functions in mammalian cells. We discuss how on the one hand regulated intramembrane proteolysis generates signaling molecules, and on the other hand how processes such as endoplasmic reticulum-associated degradation controls the quantity and activity of central regulators. While the enzymatic core of GxGD intramembrane proteases is conserved, association with regulatory factors and substrate adaptors may have tailored enzymes for various specific functions.
Assuntos
Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/enzimologia , Animais , Domínio Catalítico , Retículo Endoplasmático/metabolismo , Humanos , Filogenia , Transporte Proteico , ProteóliseRESUMO
Rhomboid intramembrane serine proteases are present in all kingdoms of life, but as we do not know their substrates in many species, it remains puzzling why rhomboids are among the most-conserved integral membrane proteins. Two new studies in The EMBO Journal by Began et al and Liu et al now link bacterial rhomboid proteases to membrane protein degradation, showing striking similarities to what is known about eukaryotic rhomboid family proteins, thus pointing toward a conserved membrane surveillance mechanism.
Assuntos
Proteínas de Bactérias , Proteínas de Membrana , ATPases Associadas a Diversas Atividades Celulares , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Degradação Associada com o Retículo Endoplasmático , Licenciamento , Proteínas de Membrana/metabolismo , Membranas/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
The endoplasmic reticulum (ER)-resident intramembrane rhomboid protease RHBDL4 generates metastable protein fragments and together with the ER-associated degradation (ERAD) machinery provides a clearance mechanism for aberrant and surplus proteins. However, the endogenous substrate spectrum and with that the role of RHBDL4 in physiological ERAD is mainly unknown. Here, we use a substrate trapping approach in combination with quantitative proteomics to identify physiological RHBDL4 substrates. This revealed oligosaccharyltransferase (OST) complex subunits such as the catalytic active subunit STT3A as substrates for the RHBDL4-dependent ERAD pathway. RHBDL4-catalysed cleavage inactivates OST subunits by triggering dislocation into the cytoplasm and subsequent proteasomal degradation. RHBDL4 thereby controls the abundance and activity of OST, suggesting a novel link between the ERAD machinery and glycosylation tuning.
Assuntos
Degradação Associada com o Retículo Endoplasmático , Hexosiltransferases , Proteínas de Membrana , Hexosiltransferases/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismoRESUMO
Over the last two decades, a group of unusual proteases, so-called intramembrane proteases, have become increasingly recognized for their unique ability to cleave peptide bonds within cellular membranes. They are found in all kingdoms of life and fulfil versatile functions ranging from protein maturation, to activation of signalling molecules, to protein degradation. In this Cell Science at a Glance article and the accompanying poster, we focus on intramembrane proteases in mammalian cells. By comparing intramembrane proteases in different cellular organelles, we set out to review their functions within the context of the roles of individual cellular compartments. Additionally, we exemplify their mode of action in relation to known substrates by distinguishing cleavage events that promote degradation of substrate from those that release active domains from the membrane bilayer.
Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Peptídeo Hidrolases/metabolismo , Proteólise , Animais , HumanosRESUMO
Tail-anchored (TA) proteins insert post-translationally into the endoplasmic reticulum (ER), the outer mitochondrial membrane (OMM) and peroxisomes. Whereas the GET pathway controls ER-targeting, no dedicated factors are known for OMM insertion, posing the question of how accuracy is achieved. The mitochondrial AAA-ATPase Msp1 removes mislocalized TA proteins from the OMM, but it is unclear, how Msp1 clients are targeted for degradation. Here we screened for factors involved in degradation of TA proteins mislocalized to mitochondria. We show that the ER-associated degradation (ERAD) E3 ubiquitin ligase Doa10 controls cytoplasmic level of Msp1 clients. Furthermore, we identified the uncharacterized OMM protein Fmp32 and the ectopically expressed subunit of the ER-mitochondria encounter structure (ERMES) complex Gem1 as native clients for Msp1 and Doa10. We propose that productive localization of TA proteins to the OMM is ensured by complex assembly, while orphan subunits are extracted by Msp1 and eventually degraded by Doa10.