The FEBS journal最新文献

Redistribution of sterols among cellular compartments is crucial for the proper functions of different organelles, but how sterols are transported in plants is barely studied. Here, we identified that Arabidopsis C2 and GRAM domain-containing proteins C2GR1/2, a specialized subgroup of the lipid transfer proteins anchored at membrane contact sites (LAMs), transport sterols between membranes via their first START-like domains (SLD1s), while the SLD2 domains are inactive. Structural studies on C2GR2-SLD1/SLD2 elucidated that the sterol transport process involves the exchange of sterol and water, which requires the proper size and the amphiphilic nature of the cavity, as well as the conformational changes of the three Ω loops at the entrance. Importantly, the amphiphilicity of the cavity is shared by other SLD domains in yeast and mammals, a feature that was overlooked by previous studies. These findings not only advance our understanding of sterol transport in plants but also redefine the sterol transport paradigm for LAM proteins.

Nucleophosmin 1 (NPM1) is a ubiquitously expressed phosphoprotein, mainly located in the nucleolus. It is overexpressed in solid tumors and considered a key target in cancer therapy. NPM1 mutations are the most common genetic abnormalities in acute myeloid leukemia (AML), where they are found in about 30% of patients. In AML, NPM1 mutations result in the cytoplasmic localization of the mutant protein NPMc+. Although NPM1 mutations are known to drive AML, the underlying mechanisms are not fully understood. In this study, we found that primary leukemia cells from NPM1-mutated AML patients exhibited elevated intracellular calcium levels compared with cells from NPM1 wild-type AML patients. Our investigation revealed that NPMc+ interacts with the calcium channel Orai1, disrupting calcium homeostasis in AML cells. Notably, we identified that the N-terminal region of NPM1 contains a calcium-binding domain that directly interacts with Orai1, facilitating calcium influx. Targeting NPMc+, Orai1, or the NPMc+/Orai1 complex using small-molecule inhibitors significantly reduced calcium influx, inhibited calcium-related signaling pathways, and suppressed the proliferation of NPM1-mutated AML cells. These findings uncover a novel mechanism in which NPMc+ interacts with Orai1, disrupting calcium homeostasis and promoting AML progression. This presents a promising therapeutic strategy targeting the NPMc+/Orai1-mediated calcium imbalance in NPM1-mutated AML.

The eukaryote-specific N-terminal domain of ribosomal protein eS31 is a flexible extension near the decoding center, but its role in translational control has been obscured by pleiotropic defects associated with complete domain deletion. To resolve this ambiguity, we dissected its function at amino acid resolution in Saccharomyces cerevisiae using a genetic screen for ribosome stalling at CGA codon repeats. By functionally separating the N-terminal ubiquitin moiety from the ribosomal domain, our screen bypassed artifacts and identified a critical cluster of basic residues (K79-K82) within this region. Charge-reversing point mutations in this cluster alleviate stalling not by impairing ribosome-associated quality control, but by reducing decoding fidelity, as supported by paromomycin sensitivity and genetic suppression by the loss of the eEF2 diphthamide modification. Crucially, the mutant lacking the entire domain operates through a fundamentally distinct mechanism, exhibiting a severe defect in termination fidelity and a paradoxical requirement for wild-type eS31 that suggests a global structural perturbation. Our findings thus resolve the function of the N-terminal domain of eS31 into two distinct modes: a residue-specific, electrostatic role in fine-tuning elongation fidelity, and a broader structural role for the entire domain in maintaining ribosome integrity for accurate termination.

Many proteins containing WW domains interact with proline-rich PPxY motifs, raising questions regarding how they achieve specificity in cellular contexts. Here, we characterize the WW domain-mediated interactions between the MAGI and IQSEC protein families, which play critical roles in neurodevelopment and synaptic signaling. The high-resolution crystal structure of the MAGI3-IQSEC3 complex reveals that an extended sequence C terminus to the canonical PPxY motif in IQSEC3 engages a previously uncharacterized binding site on the WW1 domain of MAGI3. This extension interface enhances binding affinity by dozens-fold, and mutagenesis of key residues within this site abrogates complex formation, demonstrating its functional necessity. This bipartite recognition mode is evolutionarily conserved across MAGI and IQSEC family members. Our work elucidates the structural basis governing MAGI-IQSEC assembly and establishes a generalizable model whereby motif extensions enable high-affinity, specific target selection by WW domains, with broad implications for modular domain-mediated signaling networks.

Antimicrobial resistance (AMR) has become a critical global health challenge, largely driven by metallo-β-lactamase (MBL)-mediated hydrolysis of β-lactam antibiotics, which remain the cornerstone of modern antimicrobial therapy. IMP-1, an MBL first identified in Japan, exhibits potent carbapenemase activity and currently lacks effective clinical inhibitors. To explore how distal mutations modulate catalytic behaviour in B1 MBLs, we characterised IMP-1 and its variant IMP-1-F218Y, using biophysical, biochemical, and structural approaches. Circular-dichroism spectra confirmed the preservation of the α-helical MBL fold in both enzymes, while kinetic analyses revealed enhanced hydrolysis by IMP-1-F218Y across most β-lactam substrates. Antimicrobial susceptibility-testing supported this observation, linking the increased catalytic efficiency of the mutant to elevated resistance, except under Zinc(II)-limiting conditions. The crystal structure of IMP-1-F218Y (2.9 Å; PDB ID: 8ZTB) showed an additional Y218-S262 hydrogen bond that reduces the active-site volume and stabilises the L3 loop, positioning W64 flatter across the catalytic cleft. Molecular-dynamics simulations captured this conformational compaction, indicating a more compact and catalytically favourable active site. Unlike natural IMP variants with selective substrate profiles, IMP-1-F218Y displayed an expanded substrate spectrum, demonstrating that a single distal substitution can modulate enzymatic plasticity and broaden catalytic range. These findings provide mechanistic insight into the structural adaptability of B1 MBLs and emphasise the importance of targeting such flexibility in the design of next-generation β-lactamase inhibitors.

ADAM17, from the disintegrin and metalloproteinase (ADAM) family, also called tumor necrosis factor converting enzyme (TACE), is a pleiotropic protease with more than 90 substrates, including growth factors, cytokines, receptors, and adhesion molecules. The biology of ADAM17 has been extensively studied, mainly as a regulator of epidermal growth factor receptor (EGFR) and tumor necrosis factor (TNF) signaling pathways, which are crucial for growth and inflammation, respectively. Consequently, this protease has been considered a major target to treat human cancers and inflammatory disorders. Moreover, it is involved in the pathobiology of numerous other disease types. This review summarizes the current understanding of ADAM17 and the involvement of its targets in inflammation, cancer, and cardiovascular and metabolic diseases.

Homologous recombination (HR) is generally considered dispensable in yeast and vertebrates, yet mounting evidence indicates that its essentiality depends on cellular context. Here, we dissect the basis of this context dependency in Schizosaccharomyces pombe. In the homothallic h⁹⁰ strain, regarded as wild type, mating-type switching (MTS) occurs every other cell division and requires HR to repair programmed double-strand breaks (DSBs) at the mat1 locus. We show that the widely used heterothallic h^-S strain is likewise dependent on HR for viability. HR-deficient h^-S mutants (rad51Δ, rad52Δ, or rad54Δ), still frequently employed in the literature, survive only when carrying secondary suppressor mutations that abolish mat1 DSB formation, such as smt-0, swi1Δ, or fml1Δ. In contrast, HR is dispensable in the h^+N strain, where duplication of the mat2/3 region into mat1 introduces the cenH and REIII elements. These elements nucleate H3K9 methylation and heterochromatin spreading across the imprint site, blocking imprintosome recruitment and thereby preventing both imprinting and DSB formation. Disruption of this heterochromatin, via deletion of cenH or key chromatin modifiers, restores DSB formation in h^+N cells and reinstates HR essentiality in the absence of the Clr4 methyltransferase. Collectively, our findings demonstrate that HR is indispensable for S. pombe survival due to its critical role in repairing mat1 DSBs, except under genetic or epigenetic conditions that suppress their formation.

Tissue breakdown, especially extracellular matrix or secretome breakdown is a significant aspect of physiological remodeling and disease processes in solid organs, with profound structural and regulatory impact. Tissue and circulating proteins undergo breakdown through a chemical process mediated by proteases, that is, hydrolysis of peptide bonds. Proteolysis has immense biological impact because it is irreversible, results in protein inactivation or activation, and can generate fragments with new functions to expand the functional genome. The traditional focus on a few proteases of interest against candidate substrates provided limited insights into the proteolytic landscape of human diseases. In contrast, innovations in protein terminomics workflows, tandem mass spectrometry and data handling now routinely permit identification of proteolytic events and proteases on the proteome scale, the degradome. The unbiased, de novo elucidation of disease degradomes, termed forward degradomics, has increased the number of known in vivo proteolytic events, despite only limited application to human disease to date. In reverse degradomics, activities of proteases are elucidated individually, but also at the proteome scale by digesting protein libraries sourced from tissues and cell secretomes, or by comparing the degradomes of protease-deficient/overexpressing and parental cells. Integration of forward and reverse degradomes precisely defines protease primary mechanisms in disease. Cross-disease degradome analysis can define disease-relevant, protease-specific biomarkers, identify proteases as appropriate therapeutic targets, and predict cross-organ impact of protease inhibitors. A systematic effort to map disease degradomes, that is, a prospective human degradome project would generate a comprehensive proteolysis knowledgebase for diagnostics and therapeutics.

The application of platinum-based chemotherapy is often limited by drug resistance, which involves multiple signalling pathways. Although the sulfonamide anticancer agent indisulam has been utilised as an adjuvant in combination with cisplatin, olaparib, and temozolomide in clinical trials, the mechanism by which indisulam modulates the sensitivity of gastric cancer cells to these drugs remains elusive. Here, flow cytometry, TUNEL, and CCK-8 assays demonstrated that indisulam induced apoptosis in gastric cancer cells and enhanced their sensitivity to cisplatin and oxaliplatin. Label-free quantitative proteomics identified FKBP8 as a previously undescribed downstream target of indisulam in gastric cancer cells. qPCR analysis of clinical samples revealed a strong correlation between the mRNA levels of FKBP8 and RBM39, and Kaplan-Meier plot analyses indicated that high expression of FKBP8 mRNA was associated with reduced survival time for gastric cancer patients. Mechanistically, indisulam attenuated FKBP8 transcription, and depletion of FKBP8 enhanced apoptosis and reduced colony formation in the presence of cisplatin and oxaliplatin, thereby improving the chemotherapeutic response of gastric cancer cells to these drugs. FKBP8 overexpression abrogated the effect of indisulam and cisplatin on apoptosis and cell proliferation. Experiments using a xenograft mouse model further demonstrated that the combination of indisulam and cisplatin significantly inhibited the growth of gastric cancer cells, reduced FKBP8 mRNA levels, and increased apoptosis. Taken together, this and previous studies suggest that indisulam can inhibit viability and migration, but promote apoptosis of gastric cancer cells through distinct downstream targets, suggesting that FKBP8 could be leveraged as a therapeutic target in combination with chemotherapy for gastric cancer.

The c607C>T mutation in the PACS1 gene results in an Arg203Trp substitution in the multifunctional protein PACS-1, and drives a syndrome characterized by intellectual disability, seizures, craniofacial dysmorphisms, and various characteristics of the autism spectrum. On the molecular level, this syndrome, in part, results from enhanced association of PACS-1 with the protein deacetylase HDAC6. PACS-1 uses its Furin binding region (FBR: amino acids 101-273) to directly interact with the catalytic domains of HDAC6. We present the solution structure of a chimeric PACS-1 FBR and use NMR to demonstrate that the PACS-1/HDAC6 interaction is regulated by an intramolecular mechanism involving the central unstructured region of PACS-1 folding back across the FBR and engaging in contacts with an extended, positively charged loop. The R203W substitution, located in this loop, disrupts this regulatory interaction and, in vitro, displays the ability to promote aberrant protein-protein interactions.