EMBO Journal最新文献

Neuroblastoma (NB) is the most common extracranial childhood cancer, caused by the improper differentiation of developing trunk neural crest cells (tNCC) in the sympathetic nervous system. The N⁶-methyladenosine (m⁶A) epitranscriptomic modification controls post-transcriptional gene expression but the mechanism by which the m⁶A methyltransferase complex METTL3/METTL14/WTAP is recruited to specific loci remains to be fully characterized. We explored whether the m⁶A epitranscriptome could fine-tune gene regulation in migrating/differentiating tNCC. We demonstrate that the m⁶A modification regulates the expression of HOX genes in tNCC, thereby contributing to their timely differentiation into sympathetic neurons. Furthermore, we show that posterior HOX genes are m⁶A modified in MYCN-amplified NB with reduced expression. In addition, we provide evidence that sustained overexpression of the MYCN oncogene in tNCC drives METTL3 recruitment to a specific subset of genes including posterior HOX genes creating an undifferentiated state. Moreover, METTL3 depletion/inhibition induces DNA damage and differentiation of MYCN overexpressing cells and increases vulnerability to chemotherapeutic drugs in MYCN-amplified patient-derived xenografts (PDX) in vivo, suggesting METTL3 inhibition could be a potential therapeutic approach for NB.

The mevalonate pathway produces essential lipid metabolites such as cholesterol. Although this pathway is negatively regulated by metabolic intermediates, little is known of the metabolites that positively regulate its activity. We found that the amino acid glutamine is required to activate the mevalonate pathway. Glutamine starvation inhibited cholesterol synthesis and blocked transcription of the mevalonate pathway-even in the presence of glutamine derivatives such as ammonia and α-ketoglutarate. We pinpointed this glutamine-dependent effect to a loss in the ER-to-Golgi trafficking of SCAP that licenses the activation of SREBP2, the major transcriptional regulator of cholesterol synthesis. Both enforced Golgi-to-ER retro-translocation and the expression of a nuclear SREBP2 rescued mevalonate pathway activity during glutamine starvation. In a cell model of impaired mitochondrial respiration in which glutamine uptake is enhanced, SREBP2 activation and cellular cholesterol were increased. Thus, the mevalonate pathway senses and is activated by glutamine at a previously uncharacterized step, and the modulation of glutamine synthesis may be a strategy to regulate cholesterol levels in pathophysiological conditions.

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neurotransmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and liver. We combined classical molecular dynamics simulations, Markov state modeling and hybrid first principles quantum mechanical/classical mechanical (QM/MM) simulations with experimental approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement. After isomerization to an inward-facing conformation, deprotonation of E133 and subsequent proton transfer from D46 to E133 opens the intracellular gate and permits galactonate dissociation either in its unprotonated form or after proton transfer from E133. After release of the second proton, apo DgoT returns to the outward-facing conformation. Our results provide a framework to understand how various SLC17 transport functions with distinct transport stoichiometries can be attained through subtle variations in proton and substrate binding/unbinding.

Lysosomal damage induces stress granule (SG) formation. However, the importance of SGs in determining cell fate and the precise mechanisms that mediate SG formation in response to lysosomal damage remain unclear. Here, we describe a novel calcium-dependent pathway controlling SG formation, which promotes cell survival during lysosomal damage. Mechanistically, the calcium-activated protein ALIX transduces lysosomal damage signals to SG formation by controlling eIF2α phosphorylation after sensing calcium leakage. ALIX enhances eIF2α phosphorylation by promoting the association between PKR and its activator PACT, with galectin-3 inhibiting this interaction; these regulatory events occur on damaged lysosomes. We further find that SG formation plays a crucial role in promoting cell survival upon lysosomal damage caused by factors such as SARS-CoV-2^ORF3a, adenovirus, malarial pigment, proteopathic tau, or environmental hazards. Collectively, these data provide insights into the mechanism of SG formation upon lysosomal damage and implicate it in diseases associated with damaged lysosomes and SGs.

Maturation of human mitochondrial tRNA is essential for cellular energy production, yet the underlying mechanisms remain only partially understood. Here, we present several cryo-EM structures of the mitochondrial RNase Z complex (ELAC2/SDR5C1/TRMT10C) bound to different maturation states of mitochondrial tRNA^His, showing the molecular basis for tRNA-substrate selection and catalysis. Our structural insights provide a molecular rationale for the 5'-to-3' tRNA processing order in mitochondria, the 3'-CCA antideterminant effect, and the basis for sequence-independent recognition of mitochondrial tRNA substrates. Furthermore, our study links mutations in ELAC2 to clinically relevant mitochondrial diseases, offering a deeper understanding of the molecular defects contributing to these conditions.

The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins, which are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S protein from SARS-CoV-2 variants has revealed this structural adaptation at high resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures, and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Taken together, our data are consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of the S protein.

Osmotic stress and abscisic acid (ABA) signaling are important for plant growth and abiotic stress resistance. Activation of osmotic and ABA signaling downstream of the PYL-type ABA receptors requires the release of SnRK2 protein kinases from the inhibition imposed by PP2Cs. PP2Cs are core negative regulators that constantly interact with and inhibit SnRK2s, but how osmotic signaling breaks the PP2C inhibition of SnRK2s remains unclear. Here, we report that an Arabidopsis receptor-like cytoplasmic kinase, BIK1, releases PP2C-mediated inhibition of SnRK2.6 via phosphorylation regulation. The dominant abi1-1 ABA-signaling mutation (G180D) disrupts PYL-PP2C interactions and disables PYL-initiated release of SnRK2s; in contrast, BIK1 releases abi1-1-mediated inhibition of SnRK2.6. BIK1 interacts with and phosphorylates SnRK2.6 at two tyrosine residues, which are critical for SnRK2.6 activation and function. Phosphorylation of the two tyrosine residues may affect the docking of the tryptophan "lock" of PP2C into SnRK2.6. Moreover, the bik1 mutant is defective in SnRK2 activation, stress-responsive gene expression, ABA accumulation, growth maintenance, and water loss under osmotic stress. Our findings uncover the critical role of BIK1 in releasing PP2C-mediated inhibition of SnRK2s under osmotic stress.

The class-II transactivator (CIITA) is the master regulator of MHC class-II gene expression and hence the adaptive immune response. Three cell type-specific promoters (pI, pIII, and pIV) are involved in the regulation of CIITA expression, which can be induced by IFN-γ in non-immune cells. While key regulatory elements have been identified within these promoters, our understanding of the transcription factors regulating CIITA expression is incomplete. Here, we demonstrate that the telomere-binding protein and transcriptional activator ZBTB48 directly binds to both critical activating elements within the B-cell-specific promoter CIITA pIII. ZBTB48 knockout impedes the CIITA/MHC-II expression program induced in non-APC cells by IFN-γ, and loss of ZBTB48 in mice silences MHC-II expression in pro-B and immature B cells. Transcriptional regulation of CIITA by ZBTB48 is enabled by ZBTB48-dependent chromatin opening at CIITA pIII upstream of activating H3K4me3 marks. We conclude that ZBTB48 primes CIITA pIII by acting as a molecular on-off-switch for B-cell-specific CIITA expression.

Glycosylation, which plays an important role in modifying lipids and sorting of proteins, is regulated by asymmetric intra-Golgi distribution and SPPL3-mediated cleavage of Golgi enzymes. We found that cells lacking LYSET/TMEM251, a retention factor for Golgi N-acetylglucosamine-1-phosphotransferase (GNPT), display SPPL3-dependent hypersecretion of the Golgi membrane protein B4GALT5. We demonstrate that in wild-type cells B4GALT5 is tagged with mannose 6-phosphate (M6P), a sorting tag typical of soluble lysosomal hydrolases. Hence, M6P-tagging of B4GALT5 may represent a novel degradative lysosomal pathway. We also observed B4GALT5 hypersecretion and prominent destabilization of LYSET-GNPT complexes, impaired M6P-tagging, and disturbed maturation and trafficking of lysosomal enzymes in multiple human cell lines lacking the COPI adaptors GOLPH3 and GOLPH3L. Mechanistically, we identified LYSET as a novel, atypical client of GOLPH3/GOLPH3L. Thus, by ensuring the cis-Golgi localization of the LYSET-GNPT complex and maintaining its Golgi polarity, GOLPH3/GOLPH3L is essential for the integrity of the M6P-tagging machinery and homeostasis of lysosomes.

Terminal oligopyrimidine motif-containing mRNAs (TOPs) encode all ribosomal proteins in mammals and are regulated to tune ribosome synthesis to cell state. Previous studies have implicated LARP1 in 40S- or 80S-ribosome complexes that are thought to repress and stabilize TOPs. However, a molecular understanding of how LARP1 and TOPs interact with these ribosome complexes is lacking. Here, we show that LARP1 directly binds non-translating ribosomal subunits. Cryo-EM structures reveal a previously uncharacterized domain of LARP1 bound to and occluding the mRNA channel of the 40S subunit. Increased availability of free ribosomal subunits downstream of various stresses promote 60S joining at the same interface to form LARP1-80S complexes. Simultaneously, LARP1 engages the TOP via its previously characterized La/PAM2 and DM15 domains. Contrary to expectations, ribosome binding within these complexes is not required for LARP1-mediated TOP repression or stabilization, two canonical LARP1 functions. Together, this work provides molecular insight into how LARP1 directly binds ribosomal subunits and challenges existing models describing the function of repressed LARP1-40S/80S-TOP complexes.