Journal of Biological Chemistry最新文献

Mutations in the ATP-binding cassette transporter ABCA4 are responsible for recessive Stargardt disease (STGD1) a juvenile form of macular degeneration. In preclinical and clinical studies it has been shown that deficiency in ABCA4 leads to accelerated formation of the toxic bisretinoid fluorophores that form as the product of nonenzymatic reactions of retinaldehyde with phosphatidylethanolamine (PE) (2:1 ratio). Here, by comparing photoreceptor cell viability in albino versus agouti Abca4^-/- mice and by dark-rearing albino Abca4^-/- mice we show that photoreceptor cell degeneration in the Abca4^-/- mouse is at least partially driven by light. Elevated vitamin A in chow and a high fat diet reduced photoreceptor cell viability. PE and N-retinylidiene-phosphatidylethanolamine were reduced as were steady state levels of retinoid in light-adapted eyes. As expected, bisretinoids, measured as short-wavelength fundus autofluorescence, were elevated in both pigmented and albino Abca4^-/- mice. Hyperautofluorescent puncta in fundus autofluorescence images colocalized in spectral domain optical coherence tomography scans with aberrant hyperreflectivity that occupied photoreceptor-attributable bands and extended anteriorly to interrupt the ellipsoid zone and external limiting membrane. In epifluorescence images of Abca4^-/- retina, retinal pigment epithelium was autofluorescent due to bisretinoid accumulation. Occasionally AF lesions extended anteriorly from the RPE to a horizontal band exhibiting less pronounced autofluorescence at the level of photoreceptor inner and outer segments. These lesions did not co-localize with IBA1-labeled microglia. The hyperautofluorescent foci that presented as hyperreflective lesions in SD-OCT form in photoreceptor inner segments and are reminiscent of fundus flecks in STGD1.

Many GPCRs use endocytosis to promote gene transcription by prolonging signaling through the Gs-coupled cAMP / cAMP-dependent protein kinase (PKA) cascade. However, not all GPCRs efficiently internalize after agonist-induced activation and, among those that do, considerable differences have been observed in the ability of different GPCRs to stimulate endosomal cAMP production in different cell types. We asked if endocytosis distinguishes the signaling profiles of GPCRs that are naturally coexpressed in the same cells, focusing on three Gs-coupled GPCRs endogenous to human kidney-derived (HEK293) cells: the vasoactive intestinal peptide receptor-1 (VIPR1 / VPAC1) and β2-adrenergic receptor (β₂AR / ADRB2) which both rapidly internalize after activation and the adenosine-2B receptor (A_2BR / ADORA2B) which we show here does not. For VIPR1, endocytosis significantly prolongs both the global cAMP elevation and cytoplasmic PKA activity increase. For β2AR, endocytosis has little effect on the global cAMP elevation but, nonetheless, it significantly prolongs the cytoplasmic PKA activity increase. A_2BR differs still further, with endocytosis having little effect on signal duration measured at either intermediate step. We then show that further downstream steps in the cascade, nuclear PKA activation and transcriptional induction, are significantly endocytosis-dependent when stimulated through VIPR1 and β2AR but endocytosis-independent through A_2BR. We conclude that endocytosis indeed distinguishes the signaling profiles of endogenously coexpressed GPCRs. We propose that quantitative differences in GPCR internalization and activation in endosomes program in cells a GPCR-selective, spatiotemporal 'cAMP code' that is spatially 'decoded' by proximity to local cytoplasmic PKA stores and then temporally interpreted by the nucleus.

Rho GTPases are members of the Ras superfamily of small GTPases that regulate cell morphology, motility, polarization and cell cycling. Like members of the Ras subfamily, Rho subfamily GTPases dysregulation is implicated in a range of tumors and can serve as a valid drug target. In this work, we investigate the evolutionary trajectory of Rho GTPases within a region of the protein that has been exploited for cancer drug discovery within the Ras subfamily branch - the "switch II pocket". Our previous work has illustrated the role of allostery in this region of H-Ras in modulation of intrinsic hydrolysis and effector-binding capacity. Here, we report that a highly conserved salt bridge within the Rho subfamily stabilizes the RhoA GTPase active site in a catalytically favorable conformation. We probed the roles of the Rho salt bridge via X-ray crystallography, accelerated molecular dynamics simulations (aMD), and enzymatic studies. We showed that the removal of a residue within switch II of RhoA, the salt bridge residue R70, can impart catastrophic effects on active site organization and GTP hydrolysis. As expected, removal of the analogous R68 in H-Ras, which is not involved in a salt bridge interaction, results in a structure with changes in the active site and a decrease in GTP hydrolysis rate constant that are more moderate than observed for RhoA. The anionic partner of R70, E102, also modulates active site conformation and, upon removal, decreases intrinsic hydrolysis. Based on aMD simulations, we uncovered evidence of epistatic relationships between the Rho salt bridge, the distal residue K98 and P-loop residue D13 which coordinate allosteric communication from the switch regions directly to the active site. Finally, we describe the functional landscape of switch II pocket in the context of both Rho subfamily evolution and potential for drug discovery.

The function of extracellular vesicles (EVs) is determined by the molecular cargo they carry from their parent cells. Although Apoptosis-linked gene 2-interacting protein X (Alix) is known to regulate EV cargo loading and functional properties, the specific mechanisms underlying its role in mediating β-catenin sorting and function remain unclear. In this study, we first observed the co-localization of Alix and β-catenin through immunofluorescence staining. To assess whether the interaction between Alix and β-catenin affects the function of mesenchymal stem cell (MSC)-derived EVs, we generated Alix-knockdown (KD) and Alix-overexpressing (OE) MSCs via viral transduction. Analysis of secreted EVs revealed that those derived from Alix-OE-MSCs promoted angiogenesis both in vitro and in a mouse model of hindlimb ischemia, whereas EVs from Alix-KD-MSCs suppressed angiogenesis. Mechanistically, we confirmed that the Alix-β-catenin interaction selectively enhances β-catenin enrichment within EVs. In conclusion, our findings demonstrate that Alix plays a critical role in selectively packaging β-catenin into EVs, thereby enhancing their pro-angiogenic potency. Modified.

Sialic acids are the terminal monosaccharides of the glycocalyx that critically shape cell-cell interactions, and are strongly implicated in regulating immune recognition and tissue homeostasis. In cancer, aberrant sialylation rewires the tumor microenvironment by enhancing ligands of the inhibitory Siglecs, suppressing immune effector functions, and facilitating metastatic dissemination. This review provides a comprehensive synthesis of the dual role of sialyltransferases (the "writers") and Siglecs/Selectins (the "readers") in cancer progression. We examine the structural and functional diversity of these molecules, their dysregulation in malignancy, and their impact on tumor-immune dynamics. Finally, we highlight emerging therapeutic strategies, including sialyltransferase inhibitors, sialidase conjugates, and Siglec-targeted immunotherapies, which collectively position the sialome as a tractable frontier in cancer treatment.

Due to the limited availability of metals inside the human body, pathogenic bacteria must produce multiple highly specialized metal transporters to cause infection. These transporters constitute attractive targets for developing novel antibacterial strategies. Streptococcus pyogenes possesses three iron transporters, of which the FtsABCD system is specialized in the uptake of ferric hydroxamates. The role of this transporter in infection ^{remains unclear. In this study, we developed a monoclonal alpaca V}H^{H, or nanobody, Nb1,} targeting FtsB. Nb1 binds to FtsB with sub-nM affinity, in an enthalpy-driven manner, and with a characteristically slow dissociation rate. Solvent accessibility analysis by HDX-MS, mutational analyses, and X-ray crystallography revealed that the epitope of Nb1 is in the binding pocket of FtsB. The nanobody competitively inhibited the binding of multiple hydroxamate siderophores and partially inhibited the uptake of siderophores in Streptococcus pyogenes cells. The inhibitory activity of Nb1 on siderophore transport represents a new tool to study the role of the FtsABCD transporter and can be used as a potential inhibitor of Streptococcus pyogenes growth under iron-limited conditions.

Biomolecular NMR spectroscopy has been a keystone in structural biology for decades. It can provide unique, atomic-level insights into protein dynamics, interactions, and conformational ensembles. However, its complex workflows and fragmented data analysis pipelines are often perceived as significant barriers to entry. This review highlights the POKY suite as a comprehensive solution that modernizes and streamlines the entire biomolecular NMR process. From spectral processing to structure calculation, POKY creates a single user-friendly cyberinfrastructure for a seamless and efficient NMR data analysis environment. A key aspect of its design is the integration of various artificial intelligence (AI) components to streamline complex tasks and reduce user burden, such as automation, unsupervised learning, and more. While recent advances within in silico AI prediction models have raised questions about the role of experimental data, POKY provides a clear answer. This ecosystem can create a powerful synergy between the experimental data with structure prediction. modernizing the experimental workflow, POKY makes NMR more accessible and powerful, reinforcing its vital role instructural biology.

Mechanistic studies have yielded novel prostaglandin analogs and acyclic products initially of interest in understanding cyclooxygenase structure-function, later found in vivo and of interest due to unique biological activities. Beyond arachidonic acid, fatty acid substrates span from 18 to 22 carbons and may contain ester/amide modification or epoxide/hydroxy moieties at the first double bond. Stereo control with the unconventional substrates remains largely intact although cyclization may be diverted or halted altogether, and catalysis proceeds with insertion of one, two, or three molecules of oxygen into substrates. A switch in stereochemistry at the 15-carbon occurs in a natural cyclooxygenase from coral and has received attention upon aspirin treatment of COX-2. The latter produces 15R-HETE and analogs from other fatty acids that may be further oxygenated by lipoxygenases. Functional plasticity in cyclooxygenase catalysis has enabled the formation and discovery of a host of novel eicosanoids and provided mechanistic insight into the COX reaction mechanisms.

Targeted protein degradation (TPD) has emerged as a powerful approach for eliminating disease-associated proteins by harnessing the ubiquitin-proteasome system. Biologic degraders are modular protein chimeras that recruit ubiquitin machinery to target proteins. They offer high specificity, modular design, and the ability to access targets traditionally considered challenging for small molecule ligands. This review surveys the expanding landscape of biologic TPD modalities, highlighting E3 ligase- and E2 enzyme-based degraders, TRIM-Away and TRIMbody-Away systems, and diverse biologics-based ligands that serve as target-binding components. We also discuss emerging peptide-based strategies, which bridge biologic and synthetic approaches. Finally, we highlight future opportunities to improve biologic degraders and their potential to expand the scope of targeted protein degradation.

Profiling the secretome for biomarkers offers an attractive, minimally invasive strategy to detect and monitor cancer. Several challenges, however, must be overcome including the broad dynamic range of biomolecules in the secretome and the requirement for selective detection of tumor-associated markers. Here, we employed a metabolic glycoengineering (MGE) strategy, using 1,3,4-O-Bu₃ManNAz, an azido-tagged, bioorthogonal metabolic precursor of sialic acid, to label the glycome of pancreatic near-normal and cancer cells to improve conventional LC-MS/MS proteomics-based biomarker discovery. By using this "MGE-LC-MS/MS" approach that incorporates MGE-enrichment into conventional LC-MS/MS proteomics, we identified several unique proteins from the secretomes of cancer cells evaluated in vitro. In addition to proteins known to be secreted, we identified several putatively intracellular, non-N-glycosylated proteins such β-glucocerebrosidase and paladin linked to pancreatic cancer (PC) as well as proteins associated with extracellular vesicles (EV) in PC such as DCTPP1. The identification of EV-associated proteins was consistent with our discovery that ManNAc analogs used in the MGE-LC-MS/MS workflow enhance EV production, creating a more complete secretome profile of PC cells. Pointing towards clinical relevance, we used MGE-LC-MS/MS to enrich PC-derived glycoproteins from plasma harvested from mice bearing xenografted human pancreatic tumors, unambiguously demonstrating that this approach can interrogate the secretomes of cancer cells for biomarker discovery. Finally, we discovered that MGE dramatically improved the production of EVs, which both aids in biomarker discovery (this study) and holds potential to facilitate biomanufacturing of these nascent drugs.