RESUMO
To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.
Assuntos
Proteínas de Bactérias , Microscopia Crioeletrônica , Lipídeos , Proteínas de Membrana Transportadoras , Mycobacterium tuberculosis , Internalização do Vírus , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Membrana Transportadoras/ultraestrutura , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/ultraestrutura , Tuberculose/microbiologia , Fatores de Virulência/química , Fatores de Virulência/metabolismo , Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/ultraestrutura , Periplasma/metabolismo , Domínios Proteicos , Interações Hidrofóbicas e Hidrofílicas , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/ultraestruturaRESUMO
APOEε4 is the major genetic risk factor for sporadic Alzheimer's disease (AD). Although APOEε4 is known to promote Aß pathology, recent data also support an effect of APOE polymorphism on phosphorylated Tau (pTau) pathology. To elucidate these potential effects, the pTau interactome was analyzed across APOE genotypes in the frontal cortex of 10 advanced AD cases (n = 5 APOEε3/ε3 and n = 5 APOEε4/ε4), using a combination of anti-pTau pS396/pS404 (PHF1) immunoprecipitation (IP) and mass spectrometry (MS). This proteomic approach was complemented by an analysis of anti-pTau PHF1 and anti-Aß 4G8 immunohistochemistry, performed in the frontal cortex of 21 advanced AD cases (n = 11 APOEε3/ε3 and n = 10 APOEε4/ε4). Our dataset includes 1130 and 1330 proteins enriched in IPPHF1 samples from APOEε3/ε3 and APOEε4/ε4 groups (fold change ≥ 1.50, IPPHF1 vs IPIgG ctrl). We identified 80 and 68 proteins as probable pTau interactors in APOEε3/ε3 and APOEε4/ε4 groups, respectively (SAINT score ≥ 0.80; false discovery rate (FDR) ≤ 5%). A total of 47/80 proteins were identified as more likely to interact with pTau in APOEε3/ε3 vs APOEε4/ε4 cases. Functional enrichment analyses showed that they were significantly associated with the nucleoplasm compartment and involved in RNA processing. In contrast, 35/68 proteins were identified as more likely to interact with pTau in APOEε4/ε4 vs APOEε3/ε3 cases. They were significantly associated with the synaptic compartment and involved in cellular transport. A characterization of Tau pathology in the frontal cortex showed a higher density of plaque-associated neuritic crowns, made of dystrophic axons and synapses, in APOEε4 carriers. Cerebral amyloid angiopathy was more frequent and severe in APOEε4/ε4 cases. Our study supports an influence of APOE genotype on pTau-subcellular location in AD. These results suggest a facilitation of pTau progression to Aß-affected brain regions in APOEε4 carriers, paving the way to the identification of new therapeutic targets.
Assuntos
Doença de Alzheimer , Apolipoproteína E4 , Proteínas tau , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Genótipo , Fosforilação , Proteômica , Proteínas tau/metabolismo , Proteínas tau/genéticaRESUMO
The transcription factor BACH1 regulates heme homeostasis and oxidative stress responses and promotes cancer metastasis upon aberrant accumulation. Its stability is controlled by two F-box protein ubiquitin ligases, FBXO22 and FBXL17. Here we show that the homodimeric BTB domain of BACH1 functions as a previously undescribed quaternary structure degron, which is deciphered by the two F-box proteins via distinct mechanisms. After BACH1 is released from chromatin by heme, FBXO22 asymmetrically recognizes a cross-protomer interface of the intact BACH1 BTB dimer, which is otherwise masked by the co-repressor NCOR1. If the BACH1 BTB dimer escapes the surveillance by FBXO22 due to oxidative modifications, its quaternary structure integrity is probed by a pair of FBXL17, which simultaneously engage and remodel the two BTB protomers into E3-bound monomers for ubiquitination. By unveiling the multifaceted regulatory mechanisms of BACH1 stability, our studies highlight the abilities of ubiquitin ligases to decode high-order protein assemblies and reveal therapeutic opportunities to block cancer invasion via compound-induced BACH1 destabilization.
RESUMO
Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier protein SLC25A46 interacts with both the outer and inner-membrane dynamin family GTPases Mfn1/2 and Opa1. While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with Mfn1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass-spectrometry and AlphaFold 2 modeling to identify interfaces mediating a SLC25A46 interactions with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of a Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.
RESUMO
To replicate inside human macrophages and cause the disease tuberculosis, Mycobacterium tuberculosis ( Mtb ) must scavenge a variety of nutrients from the host 1,2 . The Mammalian Cell Entry (MCE) proteins are important virulence factors in Mtb 1,3 , where they are encoded in large gene clusters and have been implicated in the transport of fatty acids 4â"7 and cholesterol 1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and how the ~10 proteins encoded in a mycobacterial mce gene cluster might assemble to transport cargo across the cell envelope remains unknown. Here we report the cryo-EM structure of the endogenous Mce1 fatty acid import machine from Mycobacterium smegmatis , a non-pathogenic relative of Mtb . The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex, long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Unexpectedly, our structural data revealed the presence of a previously unknown subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated fatty acid import across the mycobacterial cell envelope.