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Autophagy facilitates the degradation of cellular content via the lysosome and is involved in cellular homeostasis and stress response pathways. As such, malfunction of autophagy is linked to a variety of diseases ranging from organ-specific illnesses like cardiomyopathy to systemic illnesses such as cancer or metabolic syndromes. Given the variety of autophagic functions within a cell and tissue, regulation of autophagy is complex and contains numerous positive and negative feedback loops. While our knowledge of mechanisms for cargo selectivity has significantly improved over the last decade, our understanding of signaling routes activating individual autophagy pathways remains rather sparse. In this resource study, we report on a well-characterized chemical library containing 77 GPCR-targeting ligands that was used to systematically analyze LC3B-based autophagy as well as ER-phagy flux upon compound treatment. Upon others, compounds TC-G 1004, BAY 60-6583, PSNCBAM-1, TC-G 1008, LPA2 Antagonist 1, ML-154, JTC-801 and ML-290 targeting adenosine receptor A2a (ADORA2A), adenosine receptor A2b (ADORA2B), cannabinoid receptor 1 (CNR1), G-protein coupled receptor 39 (GPR39), lysophosphatidic acid receptor 2 (LPAR2), neuropeptide S receptor 1 (NPSR1), opioid related nociceptin receptor 1 (OPRL1), and relaxin receptor 1 (RXFP1), respectively, were hit compounds for general autophagy flux. From these compounds, only JTC-801 markly increased ER-phagy flux. In addition, the global impact of these selected hit compounds were analyzed by TMT-based mass spectrometry and demonstrated the differential impact of targeting GPCRs on autophagy-associated proteins. This chemical screening exercise indicates to a significant cross-talk between GPCR signaling and regulation of autophagy pathways.
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Autofagia , Receptores Acoplados a Proteínas G , Autofagia/efectos de los fármacos , Humanos , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , LigandosRESUMEN
Mitophagy is a specific type of autophagy responsible for the selective elimination of dysfunctional or superfluous mitochondria, ensuring the maintenance of mitochondrial quality control. The initiation of mitophagy is coordinated by the ULK1 kinase complex, which engages mitophagy receptors via its FIP200 subunit. Whether FIP200 performs additional functions in the subsequent later phases of mitophagy beyond this initial step and how its regulation occurs, remains unclear. Our findings reveal that multiple phosphorylation events on FIP200 differentially control the early and late stages of mitophagy. Furthermore, these phosphorylation events influence FIP200's interaction with ATG16L1. In summary, our results highlight the necessity for precise and dynamic regulation of FIP200, underscoring its importance in the progression of mitophagy.
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Proteínas Relacionadas con la Autofagia , Mitocondrias , Mitofagia , Fosforilación , Proteínas Relacionadas con la Autofagia/metabolismo , Proteínas Relacionadas con la Autofagia/genética , Humanos , Mitocondrias/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Células HeLa , Proteínas Portadoras/metabolismoRESUMEN
MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.
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MicroARNs , Elementos de Respuesta , MicroARNs/genética , MicroARNs/metabolismo , Animales , Ratones , Estabilidad del ARN/genética , Regiones no Traducidas 3'/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Linfocitos T/metabolismo , Sistemas de Lectura Abierta/genética , Ratones Endogámicos C57BLRESUMEN
Introduction: Influenza A virus (IAV) infection can cause the often-lethal acute respiratory distress syndrome (ARDS) of the lung. Concomitantly, acute kidney injury (AKI) is frequently noticed during IAV infection, correlating with an increased mortality. The aim of this study was to elucidate the interaction of IAV with human kidney cells and, thereby, to assess the mechanisms underlying IAV-mediated AKI. Methods: To investigate IAV effects on nephron cells we performed infectivity assays with human IAV, as well as with human isolates of either low or highly pathogenic avian IAV. Also, transcriptome and proteome analysis of IAV-infected primary human distal tubular kidney cells (DTC) was performed. Furthermore, the DTC transcriptome was compared to existing transcriptomic data from IAV-infected lung and trachea cells. Results: We demonstrate productive replication of all tested IAV strains on primary and immortalized nephron cells. Comparison of our transcriptome and proteome analysis of H1N1-type IAV-infected human primary distal tubular cells (DTC) with existing data from H1N1-type IAV-infected lung and primary trachea cells revealed enrichment of specific factors responsible for regulated cell death in primary DTC, which could be targeted by specific inhibitors. Discussion: IAV not only infects, but also productively replicates on different human nephron cells. Importantly, multi-omics analysis revealed regulated cell death as potential contributing factor for the clinically observed kidney pathology in influenza.
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Lesión Renal Aguda , Subtipo H1N1 del Virus de la Influenza A , Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Muerte Celular Regulada , Humanos , Proteoma/metabolismo , Subtipo H3N2 del Virus de la Influenza A/fisiología , Replicación Viral/fisiología , Riñón/patología , Infecciones por Orthomyxoviridae/patologíaRESUMEN
Heterozygous mutations in the TBK1 gene can cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The majority of TBK1-ALS/FTD patients carry deleterious loss-of-expression mutations, and it is still unclear which TBK1 function leads to neurodegeneration. We investigated the impact of the pathogenic TBK1 missense variant p.E696K, which does not abolish protein expression, but leads to a selective loss of TBK1 binding to the autophagy adaptor protein and TBK1 substrate optineurin. Using organelle-specific proteomics, we found that in a knock-in mouse model and human iPSC-derived motor neurons, the p.E696K mutation causes presymptomatic onset of autophagolysosomal dysfunction in neurons precipitating the accumulation of damaged lysosomes. This is followed by a progressive, age-dependent motor neuron disease. Contrary to the phenotype of mice with full Tbk1 knock-out, RIPK/TNF-α-dependent hepatic, neuronal necroptosis, and overt autoinflammation were not detected. Our in vivo results indicate autophagolysosomal dysfunction as a trigger for neurodegeneration and a promising therapeutic target in TBK1-ALS/FTD.
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Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/patología , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/patología , Neuronas Motoras/patología , Mutación , Enfermedades Neuroinflamatorias , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Glioblastoma is an incurable brain tumor with a median survival below two years. Trials investigating targeted therapy with inhibitors of the kinase mTOR have produced ambiguous results. Especially combination of mTOR inhibition with standard temozolomide radiochemotherapy has resulted in reduced survival in a phase II clinical trial. To date, this phenomenon is only poorly understood. To recreate the therapeutic setting in vitro, we exposed glioblastoma cell lines to co-treatment with rapamycin and temozolomide and assessed cell viability, DNA damage and reactive oxygen species. Additionally, we employed a novel translatomic based mass spectrometry approach ("mePROD") to analyze acute changes in translated proteins. mTOR inhibition with rapamycin protected glioblastoma cells from temozolomide toxicity. Following co-treatment of temozolomide with rapamycin, an increased translation of reactive oxygen species (ROS)-detoxifying proteins was detected by mass spectrometry. This was accompanied by improved ROS-homeostasis and reduced DNA damage. Additionally, rapamycin induced the expression of the DNA repair enzyme O-6-methylguanine-DNA methyltransferase (MGMT) in glioblastoma cells with an unmethylated MGMT gene promotor. Inhibition of mTOR antagonized the cytotoxic effects of temozolomide in vitro. The induction of antioxidant defences and MGMT are two underlying candidate mechanisms. Further functional experiments in vitro and in vivo are warranted to characterize this effect that appears relevant for combinatorial therapeutic strategies.
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Despite the importance of rapid adaptive responses in the course of inflammation and the notion that post-transcriptional regulation plays an important role herein, relevant translational alterations, especially during the resolution phase, remain largely elusive. In the present study, we analyzed translational changes in inflammatory bone marrow-derived macrophages upon resolution-promoting efferocytosis. Total RNA-sequencing confirmed that apoptotic cell phagocytosis induced a pro-resolution signature in LPS/IFNγ-stimulated macrophages (MÏ). While inflammation-dependent transcriptional changes were relatively small between efferocytic and non-efferocytic MÏ; considerable differences were observed at the level of de novo synthesized proteins. Interestingly, translationally regulated targets in response to inflammatory stimuli were mostly downregulated, with only minimal impact of efferocytosis. Amongst these targets, pro-resolving matrix metallopeptidase 12 (Mmp12) was identified as a translationally repressed candidate during early inflammation that recovered during the resolution phase. Functionally, reduced MMP12 production enhanced matrix-dependent migration of MÏ. Conclusively, translational control of MMP12 emerged as an efficient strategy to alter the migratory properties of MÏ throughout the inflammatory response, enabling MÏ migration within the early inflammatory phase while restricting migration during the resolution phase.
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Metaloproteinasa 12 de la Matriz , Fagocitosis , Humanos , Metaloproteinasa 12 de la Matriz/genética , Metaloproteinasa 12 de la Matriz/metabolismo , Fagocitosis/fisiología , Macrófagos/metabolismo , Inflamación/metabolismo , Regulación de la Expresión Génica , Apoptosis/fisiologíaRESUMEN
Glioblastoma is the most common primary brain cancer in adults and represents one of the worst cancer diagnoses for patients. Suffering from a poor prognosis and limited treatment options, tumor recurrences are virtually inevitable. Additionally, treatment resistance is very common for this disease and worsens the prognosis. These and other factors are hypothesized to be largely due to the fact that glioblastoma cells are known to be able to obtain stem-like traits, thereby driving these phenotypes. Recently, we have shown that the in vitro and ex vivo treatment of glioblastoma stem-like cells with the hormonally active form of vitamin D3, calcitriol (1α,25(OH)2-vitamin D3) can block stemness in a subset of cell lines and reduce tumor growth. Here, we expanded our cell panel to over 40 different cultures and can show that, while half of the tested cell lines are sensitive, a quarter can be classified as high responders. Using genetic and proteomic analysis, we further determined that treatment success can be partially explained by specific polymorphism of the vitamin D3 receptor and that high responders display a proteome suggestive of blockade of stemness, as well as migratory potential.
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The nucleolar scaffold protein NPM1 is a multifunctional regulator of cellular homeostasis, genome integrity, and stress response. NPM1 mutations, known as NPM1c variants promoting its aberrant cytoplasmic localization, are the most frequent genetic alterations in acute myeloid leukemia (AML). A hallmark of AML cells is their dependency on elevated autophagic flux. Here, we show that NPM1 and NPM1c induce the autophagy-lysosome pathway by activating the master transcription factor TFEB, thereby coordinating the expression of lysosomal proteins and autophagy regulators. Importantly, both NPM1 and NPM1c bind to autophagy modifiers of the GABARAP subfamily through an atypical binding module preserved within its N terminus. The propensity of NPM1c to induce autophagy depends on this module, likely indicating that NPM1c exerts its pro-autophagic activity by direct engagement with GABARAPL1. Our data report a non-canonical binding mode of GABARAP family members that drives the pro-autophagic potential of NPM1c, potentially enabling therapeutic options.
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Leucemia Mieloide Aguda , Proteínas Nucleares , Humanos , Proteínas Nucleares/metabolismo , Leucemia Mieloide Aguda/metabolismo , Autofagia/fisiología , Mutación/genética , Lisosomas/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismoRESUMEN
Induction of alternative, non-apoptotic cell death programs such as cell-lethal autophagy and mitophagy represent possible strategies to combat glioblastoma (GBM). Here we report that VLX600, a novel iron chelator and oxidative phosphorylation (OXPHOS) inhibitor, induces a caspase-independent type of cell death that is partially rescued in adherent U251 ATG5/7 (autophagy related 5/7) knockout (KO) GBM cells and NCH644 ATG5/7 knockdown (KD) glioma stem-like cells (GSCs), suggesting that VLX600 induces an autophagy-dependent cell death (ADCD) in GBM. This ADCD is accompanied by decreased oxygen consumption, increased expression/mitochondrial localization of BNIP3 (BCL2 interacting protein 3) and BNIP3L (BCL2 interacting protein 3 like), the induction of mitophagy as demonstrated by diminished levels of mitochondrial marker proteins [e.g., COX4I1 (cytochrome c oxidase subunit 4I1)] and the mitoKeima assay as well as increased histone H3 and H4 lysine tri-methylation. Furthermore, the extracellular addition of iron is able to significantly rescue VLX600-induced cell death and mitophagy, pointing out an important role of iron metabolism for GBM cell homeostasis. Interestingly, VLX600 is also able to completely eliminate NCH644 GSC tumors in an organotypic brain slice transplantation model. Our data support the therapeutic concept of ADCD induction in GBM and suggest that VLX600 may be an interesting novel drug candidate for the treatment of this tumor.NEW & NOTEWORTHY Induction of cell-lethal autophagy represents a possible strategy to combat glioblastoma (GBM). Here, we demonstrate that the novel iron chelator and OXPHOS inhibitor VLX600 exerts pronounced tumor cell-killing effects in adherently cultured GBM cells and glioblastoma stem-like cell (GSC) spheroid cultures that depend on the iron-chelating function of VLX600 and on autophagy activation, underscoring the context-dependent role of autophagy in therapy responses. VLX600 represents an interesting novel drug candidate for the treatment of this tumor.
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Antineoplásicos , Glioblastoma , Humanos , Mitofagia/fisiología , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Autofagia , Antineoplásicos/farmacología , Apoptosis , Proteínas Mitocondriales/metabolismo , Quelantes del Hierro/farmacología , Hierro , Proteínas Proto-Oncogénicas c-bcl-2 , Línea Celular TumoralRESUMEN
Haematopoietic stem cells (HSC) reside in the bone marrow microenvironment (BMM), where they respond to extracellular calcium [eCa2+] via the G-protein coupled calcium-sensing receptor (CaSR). Here we show that a calcium gradient exists in this BMM, and that [eCa2+] and response to [eCa2+] differ between leukaemias. CaSR influences the location of MLL-AF9+ acute myeloid leukaemia (AML) cells within this niche and differentially impacts MLL-AF9+ AML versus BCR-ABL1+ leukaemias. Deficiency of CaSR reduces AML leukaemic stem cells (LSC) 6.5-fold. CaSR interacts with filamin A, a crosslinker of actin filaments, affects stemness-associated factors and modulates pERK, ß-catenin and c-MYC signaling and intracellular levels of [Ca2+] in MLL-AF9+ AML cells. Combination treatment of cytarabine plus CaSR-inhibition in various models may be superior to cytarabine alone. Our studies suggest CaSR to be a differential and targetable factor in leukaemia progression influencing self-renewal of AML LSC via [eCa2+] cues from the BMM.
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Leucemia Mieloide Aguda , Receptores Sensibles al Calcio , Humanos , Receptores Sensibles al Calcio/genética , Proteínas Proto-Oncogénicas c-myc , Calcio , Proteínas de Fusión Oncogénica/metabolismo , Transducción de Señal , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Citarabina , Microambiente TumoralRESUMEN
Protein quality control pathways ensure a functional proteome and rely on a complex proteostasis network (PN) that is composed of molecular chaperones and proteases. Failures in the PN can lead to a broad spectrum of diseases, including neurodegenerative disorders like Alzheimer's, Parkinson's, and a range of motor neuron diseases. The EMBO workshop "Protein quality control: from molecular mechanisms to therapeutic intervention" covered all aspects of protein quality control from underlying molecular mechanisms of chaperones and proteases to stress signaling pathways and medical implications. This report summarizes the workshop and highlights selected presentations.
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Pliegue de Proteína , Proteostasis , Croacia , Chaperonas Moleculares/metabolismo , Péptido Hidrolasas/metabolismoRESUMEN
The p53 family member TAp63α plays an important role in maintaining the genetic integrity in oocytes. DNA damage, in particular DNA double strand breaks, lead to the transformation of the inhibited, only dimeric conformation into the active tetrameric one that results in the initiation of an apoptotic program. Activation requires phosphorylation by the kinase CK1 which phosphorylates TAp63α at four positions. The third phosphorylation event is the decisive step that transforms TAp63α into the active state. This third phosphorylation, however, is ~ 20 times slower than the first two phosphorylation events. This difference in the phosphorylation kinetics constitutes a safety mechanism that allows oocytes with a low degree of DNA damage to survive. So far these kinetic investigations of the phosphorylation steps have been performed with the isolated CK1 kinase domain. However, all CK1 enzymes contain C-terminal extensions that become auto-phosphorylated and inhibit the activity of the kinase. Here we have investigated the effect of auto-phosphorylation of the C-terminus in the kinase CK1δ and show that it slows down phosphorylation of the first two sites in TAp63α but basically inhibits the phosphorylation of the third site. We have identified up to ten auto-phosphorylation sites in the CK1δ C-terminal domain and show that all of them interact with the kinase domain in a "fuzzy" way in which not a single site is particularly important. Through mutation analysis we further show that hydrophobic amino acids following the phosphorylation site are important for a substrate to be able to successfully compete with the auto-inhibitory effect of the C-terminal domain. This auto-phosphorylation adds a new layer to the regulation of apoptosis in oocytes.
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Apoptosis , Daño del ADN , Fosforilación , Oocitos/metabolismo , Roturas del ADN de Doble CadenaRESUMEN
Nitro-fatty acids (NFAs) are endogenous lipid mediators causing a spectrum of anti-inflammatory effects by covalent modification of key proteins within inflammatory signaling pathways. Recent animal models of solid tumors have helped demonstrate their potential as anti-tumorigenic therapeutics. This study evaluated the anti-tumorigenic effects of NFAs in colon carcinoma cells and other solid and leukemic tumor cell lines. NFAs inhibited the ubiquitin-proteasome system (UPS) by directly targeting the 26S proteasome, leading to polyubiquitination and inhibition of the proteasome activities. UPS suppression induced the unfolded protein response, resulting in tumor cell death. The NFA-mediated effects were substantial, specific, and enduring, representing a unique mode of action for UPS suppression. This study provides mechanistic insights into the biological actions of NFAs as possible endogenous tumor-suppressive factors, indicating that NFAs might be key structures for designing a novel class of direct proteasome inhibitors.
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Complejo de la Endopetidasa Proteasomal , Ubiquitina , Animales , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Ácidos Grasos/farmacología , Inhibidores de Proteasoma/farmacologíaRESUMEN
The mitochondrial unfolded protein response (UPRmt) is essential to safeguard mitochondria from proteotoxic damage by activating a dedicated transcriptional response in the nucleus to restore proteostasis1,2. Yet, it remains unclear how the information on mitochondria misfolding stress (MMS) is signalled to the nucleus as part of the human UPRmt (refs. 3,4). Here, we show that UPRmt signalling is driven by the release of two individual signals in the cytosol-mitochondrial reactive oxygen species (mtROS) and accumulation of mitochondrial protein precursors in the cytosol (c-mtProt). Combining proteomics and genetic approaches, we identified that MMS causes the release of mtROS into the cytosol. In parallel, MMS leads to mitochondrial protein import defects causing c-mtProt accumulation. Both signals integrate to activate the UPRmt; released mtROS oxidize the cytosolic HSP40 protein DNAJA1, which leads to enhanced recruitment of cytosolic HSP70 to c-mtProt. Consequently, HSP70 releases HSF1, which translocates to the nucleus and activates transcription of UPRmt genes. Together, we identify a highly controlled cytosolic surveillance mechanism that integrates independent mitochondrial stress signals to initiate the UPRmt. These observations reveal a link between mitochondrial and cytosolic proteostasis and provide molecular insight into UPRmt signalling in human cells.
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Citosol , Mitocondrias , Estrés Proteotóxico , Respuesta de Proteína Desplegada , Humanos , Núcleo Celular/metabolismo , Citosol/metabolismo , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Respuesta de Proteína Desplegada/fisiología , Especies Reactivas de Oxígeno/metabolismo , Activación Transcripcional , Proteostasis , Estrés Proteotóxico/fisiologíaRESUMEN
SREBP2 is a master regulator of the mevalonate pathway (MVP), a biosynthetic process that drives the synthesis of dolichol, heme A, ubiquinone and cholesterol and also provides substrates for protein prenylation. Here, we identify SREBP2 as a novel substrate for USP28, a deubiquitinating enzyme that is frequently upregulated in squamous cancers. Our results show that silencing of USP28 reduces expression of MVP enzymes and lowers metabolic flux into this pathway. We also show that USP28 binds to mature SREBP2, leading to its deubiquitination and stabilisation. USP28 depletion rendered cancer cells highly sensitive to MVP inhibition by statins, which was rescued by the addition of geranyl-geranyl pyrophosphate. Analysis of human tissue microarrays revealed elevated expression of USP28, SREBP2 and MVP enzymes in lung squamous cell carcinoma (LSCC) compared to lung adenocarcinoma (LADC). Moreover, CRISPR/Cas-mediated deletion of SREBP2 selectively attenuated tumour growth in a KRas/p53/LKB1 mutant mouse model of lung cancer. Finally, we demonstrate that statins synergise with a dual USP28/25 inhibitor to reduce viability of SCC cells. Our findings suggest that combinatorial targeting of MVP and USP28 could be a therapeutic strategy for the treatment of squamous cell carcinomas.
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Carcinoma de Pulmón de Células no Pequeñas , Carcinoma de Células Escamosas , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Neoplasias Pulmonares , Ratones , Animales , Humanos , Ácido Mevalónico/metabolismo , Neoplasias Pulmonares/genética , Carcinoma de Células Escamosas/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
Acute lysosomal membrane damage reduces the cellular population of functional lysosomes. However, these damaged lysosomes have a remarkable recovery potential independent of lysosomal biogenesis and remain unaffected in cells depleted in TFEB and TFE3. We combined proximity-labelling-based proteomics, biochemistry and high-resolution microscopy to unravel a lysosomal membrane regeneration pathway that depends on ATG8, the lysosomal membrane protein LIMP2, the RAB7 GTPase-activating protein TBC1D15 and proteins required for autophagic lysosomal reformation, including dynamin-2, kinesin-5B and clathrin. Following lysosomal damage, LIMP2 acts as a lysophagy receptor to bind ATG8, which in turn recruits TBC1D15 to damaged membranes. TBC1D15 interacts with ATG8 proteins on damaged lysosomes and provides a scaffold to assemble and stabilize the autophagic lysosomal reformation machinery. This potentiates the formation of lysosomal tubules and subsequent dynamin-2-dependent scission. TBC1D15-mediated lysosome regeneration was also observed in a cell culture model of oxalate nephropathy.
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Autofagia , Dinamina II , Dinamina II/metabolismo , Membranas Intracelulares/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Lisosomas/metabolismoRESUMEN
Mitochondria are essential for cellular functions such as metabolism and apoptosis. They dynamically adapt to the changing environmental demands by adjusting their protein, nucleic acid, metabolite, and lipid contents. In addition, the mitochondrial components are modulated on different levels in response to changes, including abundance, activity, and interaction. A wide range of omics-based approaches has been developed to be able to explore mitochondrial adaptation and how mitochondrial function is compromised in disease contexts. Here, we provide an overview of the omics methods that allow us to systematically investigate the different aspects of mitochondrial biology. In addition, we show examples of how these methods have provided new biological insights. The emerging use of these toolboxes provides a more comprehensive understanding of the processes underlying mitochondrial function.
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Aclimatación , Mitocondrias , Mitocondrias/genética , Mitocondrias/metabolismo , BiologíaRESUMEN
The ancestral SARS-CoV-2 strain that initiated the Covid-19 pandemic at the end of 2019 has rapidly mutated into multiple variants of concern with variable pathogenicity and increasing immune escape strategies. However, differences in host cellular antiviral responses upon infection with SARS-CoV-2 variants remain elusive. Leveraging whole-cell proteomics, we determined host signaling pathways that are differentially modulated upon infection with the clinical isolates of the ancestral SARS-CoV-2 B.1 and the variants of concern Delta and Omicron BA.1. Our findings illustrate alterations in the global host proteome landscape upon infection with SARS-CoV-2 variants and the resulting host immune responses. Additionally, viral proteome kinetics reveal declining levels of viral protein expression during Omicron BA.1 infection when compared to ancestral B.1 and Delta variants, consistent with its reduced replication rates. Moreover, molecular assays reveal deferral activation of specific host antiviral signaling upon Omicron BA.1 and BA.2 infections. Our study provides an overview of host proteome profile of multiple SARS-CoV-2 variants and brings forth a better understanding of the instigation of key immune signaling pathways causative for the differential pathogenicity of SARS-CoV-2 variants.
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COVID-19 , SARS-CoV-2 , Humanos , Proteoma , Pandemias , Antivirales , Anticuerpos NeutralizantesRESUMEN
Autophagy is a catabolic process during which cytosolic material is enwrapped in a newly formed double-membrane structure called the autophagosome, and subsequently targeted for degradation in the lytic compartment of the cell. The fusion of autophagosomes with the lytic compartment is a tightly regulated step and involves membrane-bound SNARE proteins. These play a crucial role as they promote lipid mixing and fusion of the opposing membranes. Among the SNARE proteins implicated in autophagy, the essential SNARE protein YKT6 is the only SNARE protein that is evolutionarily conserved from yeast to humans. Here, we show that alterations in YKT6 function, in both mammalian cells and nematodes, produce early and late autophagy defects that result in reduced survival. Moreover, mammalian autophagosomal YKT6 is phospho-regulated by the ULK1 kinase, preventing premature bundling with the lysosomal SNARE proteins and thereby inhibiting autophagosome-lysosome fusion. Together, our findings reveal that timely regulation of the YKT6 phosphorylation status is crucial throughout autophagy progression and cell survival.