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Time-resolved direct observations of proteins in action provide essential mechanistic insights into biological processes. Here, we present mechanisms of action of protein disulfide isomerase (PDI)—the most versatile disulfide-introducing enzyme in the endoplasmic reticulum—during the catalysis of oxidative protein folding. Single-molecule analysis by high-speed atomic force microscopy revealed that oxidized PDI is in rapid equilibrium between open and closed conformations, whereas reduced PDI is maintained in the closed state. In the presence of unfolded substrates, oxidized PDI, but not reduced PDI, assembles to form a face-to-face dimer, creating a central hydrophobic cavity with multiple redox-active sites, where substrates are likely accommodated to undergo accelerated oxidative folding. Such PDI dimers are diverse in shape and have different lifetimes depending on substrates. To effectively guide proper oxidative protein folding, PDI regulates conformational dynamics and oligomeric states in accordance with its own redox state and the configurations or folding states of substrates. Single-molecule analysis by high-speed atomic force microscopy reveals that oxidized protein disulfide isomerase adopts a dynamic conformation in the absence of substrates and forms face-to-face dimers to accelerate oxidative folding in the presence of substrates.

Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum that is up-regulated in mouse models of, and brains of patients with, neurodegenerative diseases involving protein misfolding. PDI’s role in these diseases, however, is not fully understood. Here, we report the discovery of a reversible, neuroprotective lead optimized compound (LOC)14, that acts as a modulator of PDI. LOC14 was identified using a high-throughput screen of ∼10,000 lead-optimized compounds for potent rescue of viability of PC12 cells expressing mutant huntingtin protein, followed by an evaluation of compounds on PDI reductase activity in an in vitro screen. Isothermal titration calorimetry and fluorescence experiments revealed that binding to PDI was reversible with a K d of 62 nM, suggesting LOC14 to be the most potent PDI inhibitor reported to date. Using 2D heteronuclear single quantum correlation NMR experiments, we were able to map the binding site of LOC14 as being adjacent to the active site and to observe that binding of LOC14 forces PDI to adopt an oxidized conformation. Furthermore, we found that LOC14-induced oxidation of PDI has a neuroprotective effect not only in cell culture, but also in corticostriatal brain slice cultures. LOC14 exhibited high stability in mouse liver microsomes and blood plasma, low intrinsic microsome clearance, and low plasma-protein binding. These results suggest that LOC14 is a promising lead compound to evaluate the potential therapeutic effects of modulating PDI in animal models of disease. Significance Protein disulfide isomerase (PDI) is a chaperone protein in the endoplasmic reticulum. It is up-regulated in mouse models of, and brains of patients with, neurological protein folding diseases. Irreversible inhibition of PDI activity by the small molecule 16F16 results in protection in cell and organotypic brain slice culture models of Huntington disease. Here, we identified lead optimized compound (LOC)14 as a nanomolar, reversible inhibitor of PDI that protects PC12 cells and medium spiny neurons from the toxic mutant huntingtin protein. LOC14 has improved potency compared with 16F16 and displays favorable pharmaceutical properties, making it a suitable compound to evaluate the therapeutic potential of inhibiting PDI in multiple disease models.

Electron cryo-microscopy structures of the human peptide-loading complex shed light on its operation and on the onset of adaptive immune responses. Structure of a peptide loader The peptide-loading complex (PLC) is a dynamic membrane complex in the endoplasmic reticulum that regulates the transport and loading of antigenic peptides onto major histocompatibility complex class I (MHC-I) molecules. As such, this complex has a key role in important adaptive immune responses to infections and tumour progression. Here, Robert Tampé and colleagues report the structure of the human PLC by electron cryo-microscopy. The editing modules of the complex are centred around the TAP transporter, which delivers the peptides from the cytosol, and peptide loading appears to induce changes in the structure of MHC-I, releasing the stable peptide/MHC-I complexes from the PLC. This provides glimpses into the mechanism of the PLC, antigen processing and the onset of MHC-I-mediated immunity. The peptide-loading complex (PLC) is a transient, multisubunit membrane complex in the endoplasmic reticulum that is essential for establishing a hierarchical immune response. The PLC coordinates peptide translocation into the endoplasmic reticulum with loading and editing of major histocompatibility complex class I (MHC-I) molecules. After final proofreading in the PLC, stable peptide–MHC-I complexes are released to the cell surface to evoke a T-cell response against infected or malignant cells 1 , 2 . Sampling of different MHC-I allomorphs requires the precise coordination of seven different subunits in a single macromolecular assembly, including the transporter associated with antigen processing (TAP1 and TAP2, jointly referred to as TAP), the oxidoreductase ERp57, the MHC-I heterodimer, and the chaperones tapasin and calreticulin 3 , 4 . The molecular organization of and mechanistic events that take place in the PLC are unknown owing to the heterogeneous composition and intrinsically dynamic nature of the complex. Here, we isolate human PLC from Burkitt’s lymphoma cells using an engineered viral inhibitor as bait and determine the structure of native PLC by electron cryo-microscopy. Two endoplasmic reticulum-resident editing modules composed of tapasin, calreticulin, ERp57, and MHC-I are centred around TAP in a pseudo-symmetric orientation. A multivalent chaperone network within and across the editing modules establishes the proofreading function at two lateral binding platforms for MHC-I molecules. The lectin-like domain of calreticulin senses the MHC-I glycan, whereas the P domain reaches over the MHC-I peptide-binding pocket towards ERp57. This arrangement allows tapasin to facilitate peptide editing by clamping MHC-I. The translocation pathway of TAP opens out into a large endoplasmic reticulum lumenal cavity, confined by the membrane entry points of tapasin and MHC-I. Two lateral windows channel the antigenic peptides to MHC-I. Structures of PLC captured at distinct assembly states provide mechanistic insight into the recruitment and release of MHC-I. Our work defines the molecular symbiosis of an ABC transporter and an endoplasmic reticulum chaperone network in MHC-I assembly and provides insight into the onset of the adaptive immune response.

Complicated and sophisticated protein homeostasis (proteostasis) networks in the endoplasmic reticulum (ER), comprising disulfide catalysts, molecular chaperones, and their regulators, help to maintain cell viability. Newly synthesized proteins inserted into the ER need to fold and assemble into unique native structures to fulfill their physiological functions, and this is assisted by protein disulfide isomerase (PDI) family. Herein, we focus on recent advances in understanding the detailed mechanisms of PDI family members as guides for client folding and assembly to ensure the efficient production of secretory proteins.

Disulfide bonds play a pivotal role in maintaining the natural structures of proteins to ensure their performance of normal biological functions. Moreover, biological molecular assembly, such as the gluten network, is also largely dependent on the intermolecular crosslinking via disulfide bonds. In eukaryotes, the formation and rearrangement of most intra- and intermolecular disulfide bonds in the endoplasmic reticulum (ER) are mediated by protein disulfide isomerases (PDIs), which consist of multiple thioredoxin-like domains. These domains assist correct folding of proteins, as well as effectively prevent the aggregation of misfolded ones. Protein misfolding often leads to the formation of pathological protein aggregations that cause many diseases. On the other hand, glutenin aggregation and subsequent crosslinking are required for the formation of a rheologically dominating gluten network. Herein, the mechanism of PDI-regulated disulfide bond formation is important for understanding not only protein folding and associated diseases, but also the formation of functional biomolecular assembly. This review systematically illustrated the process of human protein disulfide isomerase (hPDI) mediated disulfide bond formation and complemented this with the current mechanism of wheat protein disulfide isomerase (wPDI) catalyzed formation of gluten networks.

The mammalian endoplasmic reticulum (ER) harbors disulfide bond-generating enzymes, including Ero1α and peroxiredoxin 4 (Prx4) and nearly 20 members of the protein disulfide isomerase family (PDIs), which together constitute a suitable environment for oxidative protein folding. Here, we clarified the Prx4 preferential recognition of two PDI family proteins, P5 and ERp46 and the mode of interaction between Prx4 and P5 thioredoxin domain. Detailed analyses of oxidative folding catalyzed by the reconstituted Prx4–PDIs pathways demonstrated that, while P5 and ERp46 are dedicated to rapid, but promiscuous, disulfide introduction, PDI is an efficient proofreader of non-native disulfides. Remarkably, the Prx4-dependent formation of native disulfide bonds was accelerated when PDI was combined with ERp46 or P5, suggesting that PDIs work synergistically to increase the rate and fidelity of oxidative protein folding. Thus, the mammalian ER seems to contain highly systematized oxidative networks for the efficient production of large quantities of secretory proteins.

Main conclusion Overexpression or silencing of the SlPDI could increase plants resistance or sensitivity to TYLCV through enhancing or reducing the plant’s antioxidant capacity. Tomato yellow leaf curl virus (TYLCV), a plant virus that could infect a variety of crops, is particularly destructive to tomato growth. Protein disulfide isomerase (PDI) is a member of the thioredoxin (Trx) superfamily, is capable of catalyzing the formation and heterogeneity of protein disulfide bonds and inhibiting the aggregation of misfolded proteins. Studies have shown that PDI plays important roles in plant response to abiotic stress, there is no research report on the function of PDI in response to biotic stress, especially TYLCV infection. Here, we identified a tomato PDI gene, SlPDI , was involved in regulating tomato plants resistance to TYLCV. Subcellular localization results showed that SlPDI was located at the endoplasmic reticulum (ER), and its location remained unchanged after infection with TYLCV virus. Overexpression or silencing of SlPDI could increase plants resistance or sensitivity to TYLCV. Transgenic plants that overexpressing SlPDI exhibit enhanced antioxidant activity evidenced by lower hydrogen peroxide (H 2 O 2 ) level and higher activity of superoxide dismutase (SOD) and peroxidase (POD) in comparison with WT plants, after infected by TYLCV. Moreover, the SlPDI- silencing plants showed opposite results. The promoter analyzes result showed that SlPDI was involved in response to salicylic acid (SA), and our experimental results also showed that the expression level of SlPDI was induced by SA. Taken together, our results indicated that SlPDI could regulate plant resistance to TYLCV through enhancing the protein folding function of ER and promoting the synthesis and conformation of antioxidant-related proteins.

The aim of this study was to investigate the effects and the mechanism of diosgenin, a famous plant-derived steroidal sapogenin, on memory deficits in Alzheimer's disease (AD) model mice. Diosgenin-treated 5XFAD mice exhibited significantly improved performance of object recognition memory. Diosgenin treatment significantly reduced amyloid plaques and neurofibrillary tangles in the cerebral cortex and hippocampus. Degenerated axons and presynaptic terminals that were only observed in regions closely associated with amyloid plaques were significantly reduced by diosgenin treatment. The 1,25D 3 -membrane-associated, rapid response steroid-binding protein (1,25D 3 -MARRS) was shown to be a target of diosgenin. 1,25D 3 -MARRS knockdown completely inhibited diosgenin-induced axonal growth in cortical neurons. Treatment with a neutralizing antibody against 1,25D 3 -MARRS diminished the axonal regeneration effect of diosgenin in Aβ(1–42)-induced axonal atrophy. This is the first study to demonstrate that the exogenous stimulator diosgenin activates the 1,25D 3 -MARRS pathway, which may be a very critical signaling target for anti-AD therapy.

PDIA3 is a pleiotropic protein primarily located in the endoplasmic reticulum where it is involved in protein folding, catalyzing the formation, breakage, and rearrangement of disulfide bonds. PDIA3 is implicated in numerous pathologies such as cancer, inflammation, and neurodegeneration. Although punicalagin has been proven to be a highly promising PDIA3 inhibitor and can be used as target protein in glioblastoma, it does not have sufficient selectivity for PDIA3 and is a quite-large molecule. With the aim of finding punicalagin derivatives with a simplified structure, we selected punicalin, which lacks the hexahydroxy-diphenic acid moiety. Previous docking studies suggest that this part of the molecule is not involved in the binding with PDIA3. In this study we compared the ability of punicalin to bind and inhibit PDIA3 and PDIA1. Tryptophan fluorescence quenching and disulfide reductase activity (using both glutathione and insulin as substrates) were evaluated, demonstrating the ability of punicalin to bind and inhibit PDIA3 even to a lesser extent compared to punicalagin. On the other hand, punicalin showed a very low inhibition activity towards PDIA1, demonstrating a higher selectivity for PDIA3. Protein thermal shift assay evidenced that both proteins can be destabilized by punicalin as well as punicalagin, with PDIA3 much more sensitive. Additionally, punicalin showed a higher change in the thermal stability of PDIA3, with a shift up to 8 °C. This result could explain the presence of PDIA3 aggregates, evidenced by immunofluorescence analysis, that accumulate within treated cells and that are more evident in the presence of punicalin. The results here obtained show punicalin is able to bind both proteins but with a higher selectivity for PDIA3, suggesting the possibility of developing new molecules with a simplified structure that are still able to selectively bind and inhibit PDIA3.

Uropathogenic Escherichia coli attach to tissues using pili type 1. Each pilus is composed by thousands of coiled FimA domains followed by the domains of the tip fibrillum, FimF-FimG-FimH. The domains are linked by non-covalent β-strands that must resist mechanical forces during attachment. Here, we use single-molecule force spectroscopy to measure the mechanical contribution of each domain to the stability of the pilus and monitor the oxidative folding mechanism of a single Fim domain assisted by periplasmic FimC and the oxidoreductase DsbA. We demonstrate that pilus domains bear high mechanical stability following a hierarchy by which domains close to the tip are weaker than those close to or at the pilus rod. During folding, this remarkable stability is achieved by the intervention of DsbA that not only forms strategic disulfide bonds but also serves as a chaperone assisting the folding of the domains. The pilus type 1 of uropathogenic E. coli must resist mechanical forces to remain attached to the epithelium. Here the authors use single-molecule force spectroscopy to demonstrate a hierarchy of mechanical stability among the pilus domains and show that the oxidoreductase DsbA also acts as a folding chaperone on the domains.