Journal of Biological Chemistry最新文献

Type VI secretion systems (T6SS) are dynamic protein nanomachines found in Gram-negative bacteria that deliver toxic effector proteins into target cells in a contact-dependent manner. Prior to secretion, many T6SS effector proteins require chaperones and/or accessory proteins for proper loading onto the structural components of the T6SS apparatus. However, despite their established importance, the precise molecular function of several T6SS accessory protein families remains unclear. In this study, we set out to characterize the DUF2169 family of T6SS accessory proteins. Using gene co-occurrence analyses, we find that DUF2169-encoding genes strictly co-occur with genes encoding T6SS spike complexes formed by VgrG and 'PAAR-like' DUF4150 domains. Although structural similar to PAAR domains, DUF4150 domains lack PAAR motifs and instead contain a conserved PIPY motif, leading us to designate them PIPY domains. Next, we present both genetic and biochemical evidence that PIPY domains require a cognate DUF2169 protein to form a functional T6SS spike complex with VgrG. This contrasts with canonical PAAR proteins, which bind VgrG on their own to form functional spike complexes. By solving the first crystal structure of a DUF2169 protein, we show that this T6SS accessory protein adopts a novel protein fold. Furthermore, biophysical and structural modeling data suggest that DUF2169 contains a dynamic loop that physically interacts with a hydrophobic patch on the surface of its cognate PIPY domain. Based on these findings, we propose a model whereby DUF2169 proteins function as molecular chaperones that maintain VgrG-PIPY spike complexes in a secretion-competent state prior to their export by the T6SS apparatus.

Protein mycoloylation is a newly characterized post-translational modification (PTM) specifically found in Corynebacteriales, an order of bacteria that includes numerous human pathogens. Their envelope is composed of a unique outer membrane, the so-called mycomembrane made of very-long chain fatty acids, named mycolic acids. Recently, some mycomembrane proteins including PorA have been unambiguously shown to be covalently modified with mycolic acids in the model organism Corynebacterium glutamicum by a mechanism that relies on the mycoloyltransferase MytC. This PTM represents the first example of protein O-acylation in prokaryotes and the first example of protein modification by mycolic acid. Through the design and synthesis of trehalose monomycolate (TMM) analogs, we prove that i) MytC is the mycoloyltransferase directly involved in this PTM, ii) TMM, but not trehalose dimycolate (TDM), is a suitable mycolate donor for PorA mycoloylation, iii) MytC is able to discriminate between an acyl and a mycoloyl chain in vitro unlike other trehalose mycoloyltransferases. We also solved the structure of MytC acyl-enzyme obtained with a soluble short TMM analogs which constitutes the first mycoloyltransferase structure with a covalently linked to an authentic mycolic acid moiety. These data highlight the great conformational flexibility of the active site of MytC during the reaction cycle and pave the way for a better understanding of the catalytic mechanism of all members of the mycoloyltransferase family including the essential Antigen85 enzymes in Mycobacteria.

The assembly of tau into filaments defines tauopathies, a group of neurodegenerative diseases including Alzheimer's disease (AD), Pick's disease (PiD), corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP). The seeded aggregation of tau has been modelled in cell culture using pro-aggregant modifications such as truncation of N- and C-termini and point-mutations within the microtubule-binding repeat domain. This limits the applicability of research findings to sporadic disease, where aggregates contain wild-type, full-length tau. We aimed to develop sensitive and specific biosensor assays for brain-derived tau species utilizing 0N3R and 0N4R tau expressed in HEK293 cells. We further generate a cell line that propagates insoluble tau which is hyperphosphorylated at disease relevant sites and retains a seeding profile similar to AD. We propose these systems as an advance over existing cell-based seeding assays, as they display specificity to the conformation and isoform composition of sporadic human disease.

Aberrant activation of the hedgehog (Hh) signaling pathway positively correlates with progression, invasion and metastasis of several cancers, including breast cancer. Although numerous inhibitors of the Hh signaling pathway are available, several oncogenic mutations of key components of the pathway, including Smoothened (Smo), have limited their capability to be developed as putative anti-cancer drugs. In this study, we have modulated the Hh signaling pathway in breast cancer using a specific FDA-approved phosphodiesterase 4 (PDE4) inhibitor rolipram. The results indicated that increased levels of cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA), due to the treatment with rolipram on MCF-7 and MDA-MB-231 cells, induced PKA-mediated ubiquitination of glioma-associated oncogene homolog 2 full length (GLI2FL) and GLI3FL, leading to their transformation to respective repressor forms. This in turn reduced the level of GLI1 transcription factor in a time-dependent manner. We have also shown that elevated levels of PKA reduced the level of phosphorylated glycogen synthase kinase 3β (GSK3β), which is known to augment PKA-mediated ubiquitination of GLI2FL and GLI3FL. Rolipram treatment also impaired wound healing and migration in both cell lines and significantly reduced tumor weight and volume in tumor-bearing mice. Histological analysis showed a reduction in multi-nucleated cells and cellular infiltration in the lungs of rolipram-treated mice. Moreover, rolipram decreased GLI1 levels in tumors by enhancing cAMP-PKA signaling. These findings suggest that rolipram effectively inhibits the Hh pathway downstream of Smo, offering potential as a therapeutic strategy for controlling breast cancer progression and metastasis, including both hormone-responsive and triple-negative subtypes.

MarE, a heme-dependent enzyme, catalyzes a unique 2-oxindole-forming monooxygenation reaction from tryptophan metabolites. To elucidate its enzyme-substrate interaction mode, we present the first X-ray crystal structures of MarE in complex with its prime substrate, (2S,3S)-β-methyl-L-tryptophan and cyanide at 1.89 Å resolution as well as a truncated yet catalytically active version in complex with the substrate at 2.45 Å resolution. These structures establish MarE as a member of the heme-dependent aromatic oxygenase (HDAO) superfamily and reveal its evolutionary link to indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO). While MarE adopts a global structure resembling the homotetrameric TDO, it features a simplified α6 helix compared to TDO's more elaborate αE and αH helices with additional αF and αG regions. Despite differing oxygen activation outcomes, MarE shares a substrate binding mode similar to IDO and TDO, with the indole nitrogen of its substrate oriented toward the heme iron in the ternary cyano complex, interacting with His55. The substrate's carboxylate group engages Arg118, with mutational studies confirming the roles of these residues in substrate binding. However, the second-sphere interactions with the substrate's α-amino nitrogen differ between MarE and TDO, and the substrate's orientation in the binary complex remains ambiguous due to two possible conformations. Notably, TDO features an extensive hydrogen-bonding network around the heme propionate below the heme plane, which is absent in MarE, suggesting mechanistic differences. These structural insights lay a foundation for further mechanistic studies, particularly for understanding how heme-dependent enzymes oxygenate tryptophan-derived metabolites.

Apolipoprotein E (APOE) is distributed across various human tissues and plays a crucial role in lipid metabolism. Recent investigations have uncovered an additional facet of APOE's functionality, revealing its role in host defense against bacterial infections. To assess the antibacterial attributes of APOE3 and APOE4, we conducted antibacterial assays using P. aeruginosa and E. coli. Exploring the interaction between APOE isoforms and lipopolysaccharides (LPS) from E. coli, we conducted several experiments, including gel shift assays, circular dichroism, and fluorescence spectroscopy. Furthermore, the interaction between APOE isoforms and LPS was further substantiated through atomic resolution molecular dynamics (MD) simulations. The presence of LPS induced the aggregation of APOE isoforms, a phenomenon confirmed through specific amyloid staining, as well as fluorescence and electron microscopy. The scavenging effects of APOE3/4 isoforms were studied through both in vitro and in vivo experiments. In summary, our study established that APOE isoforms exhibit binding to LPS, with a more pronounced affinity and complex formation observed for APOE4 compared to APOE3. Furthermore, our data suggest that APOE isoforms neutralize LPS through aggregation, leading to a reduction of local inflammation in experimental animal models. Additionally, both isoforms demonstrated inhibitory effects on the growth of P. aeruginosa and E. coli. These findings provide new insights into the multifunctionality of APOE in the human body, particularly its role in innate immunity during bacterial infections.

The Shab family voltage-gated K⁺ channels (i.e., Kv2.1, Kv2.2) are widely expressed in mammalian brain, and regulate neuronal action-potential firing. In addition to their canonical functions, the Kv2 proteins help establish direct attachments between plasma membrane (PM) and endoplasmic reticulum (ER), also known as ER-PM junctions. However, the biochemical properties and molecular organization of these ion-channels have not yet been described in human neurons. Here, we have performed a systematic analysis of endogenous expression, post-translational modification (PTM), and subcellular distribution of the major components of Kv2 complex in neurons derived from human stem cells. We found that both Kv2.1, Kv2.2, and their auxiliary subunit AMIGO1, are significantly upregulated during early neurogenesis, localize at cell surface, and already begin to assemble with each other. Human Kv2.1 and AMIGO1, but not Kv2.2 undergo substantial PTM including phosphorylation and/or N-linked glycosylation. Acute pharmacological inhibition with Kv2 blockers also revealed their functional activation in human neurons. These proteins formed prominent clusters at cell bodies, dendritic branches, and axon initial segments. Interestingly, a large fraction of them also exhibited considerable accumulation at human presynaptic terminals, where they aggregated with local ER network. This synaptic localization of Kv2 subunits was primarily restricted to presynaptic region, as they demonstrated limited enrichment at postsynaptic density. These results were highly reproducible in multiple stem cell lines used and alternative differentiation protocols tested, confirming that human presynaptic compartments can actively recruit the Shab family K⁺ ion-channels.

Gastric cancer (GC) remains a significant global health challenge, particularly due to the resistance of gastric cancer stem cells (GCSCs) to chemotherapy. This study investigates the role of heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), a member of the heterogeneous nuclear ribonucleoproteins (hnRNPs), in modulating mitochondrial metabolic reprogramming and contributing to chemoresistance in GCSCs. Through extensive analysis of tumor cancer genome atlas (TCGA) and gene expression omnibus (GEO) datasets, HNRNPA2B1 was identified as a key regulator in GCSCs, correlating with poor prognosis and enhanced resistance to chemoresistance. CRISPR-Cas9 mediated knockout of HNRNPA2B1 in GCSCs led to a significant decrease in mitochondrial function, reduced migration, invasion, and sphere formation abilities, and markedly increased apoptosis. These changes were accompanied by a shift in metabolic activity, evidenced by decreased oxygen consumption and increased extracellular acidification. Our results highlight HNRNPA2B1 as a pivotal factor in sustaining the malignant phenotype of GCSCs and present it as a potential therapeutic target to improve chemotherapy efficacy in GC.

Given the pivotal role of the Eph-Ephrin signaling pathway in tumor progression, agonists or antagonists targeting Eph/Ephrin have emerged as promising anticancer strategies. However, the implications of glycosylation modifications within Eph/Ephrin and their targeted protein therapeutics remain elusive. Here, we identify that N-glycosylation within the receptor-binding domain (RBD) of ephrin B1 (EFNB1) is indispensable for its functional repertoire. Notably, compared to wild-type EFNB1, the glycosylation-deficient N139D mutant drastically diminishes the sensitivity of tumor cells to chemotherapeutic agents, suggesting the existence of both glycosylation-dependent and independent effects mediated by EFNB1. Transcriptomic analysis highlights immune response and oxidative phosphorylation as the primary signaling pathways modulated by glycosylation modifications. In coculture systems, the EFNB1-RBD-Fc recombinant protein, while inhibiting B-lymphoma cells, also exerts differential impacts on stromal cells depending on their glycosylation status. Furthermore, the efficacy of both glycosylated and non-glycosylated EFNB1-RBD-Fc is influenced by the endogenous EFNB1 levels within tumor cells. Taking together, this study demonstrates the complexity and multifaceted roles of glycosylation in modulating EFNB1 function. These findings underscore the need for a nuanced understanding of glycosylation patterns in Eph/Ephrin-targeted therapies to optimize their therapeutic potential.