Termini restraining of small membrane proteins enables structure determination at near-atomic resolution

Small membrane proteins are difficult targets for structural characterization. Here, we stabilize their folding by restraining their amino and carboxyl termini with associable protein entities, exemplified by the two halves of a superfolder GFP. The termini-restrained proteins are functional and show improved stability during overexpression and purification. The reassembled GFP provides a versatile scaffold for membrane protein crystallization, enables diffraction to atomic resolution, and facilitates crystal identification, phase determination, and density modification. This strategy gives rise to 14 new structures of five vertebrate proteins from distinct functional families, bringing a substantial expansion to the structural database of small membrane proteins. Moreover, a high-resolution structure of bacterial DsbB reveals that this thiol oxidoreductase is activated through a catalytic triad, similar to cysteine proteases. Overall, termini restraining proves exceptionally effective for stabilization and structure determination of small membrane proteins.

What Role Does COA6 Play in Cytochrome C Oxidase Biogenesis: A Metallochaperone or Thiol Oxidoreductase, or Both?

Complex IV (cytochrome c oxidase; COX) is the terminal complex of the mitochondrial electron transport chain. Copper is essential for COX assembly, activity, and stability, and is incorporated into the dinuclear CuA and mononuclear CuB sites. Multiple assembly factors play roles in the biogenesis of these sites within COX and the failure of this intricate process, such as through mutations to these factors, disrupts COX assembly and activity. Various studies over the last ten years have revealed that the assembly factor COA6, a small intermembrane space-located protein with a twin CX9C motif, plays a role in the biogenesis of the CuA site. However, how COA6 and its copper binding properties contribute to the assembly of this site has been a controversial area of research. In this review, we summarize our current understanding of the molecular mechanisms by which COA6 participates in COX biogenesis.

Tumor cells rely on the thiol oxidoreductase PDI for PERK signaling in order to survive ER stress

Upon ER stress cells activate the unfolded protein response through PERK, IRE1 and ATF6. Remarkable effort has been made to delineate the downstream signaling of these three ER stress sensors after activation, but upstream regulation at the ER luminal site still remains mostly undefined. Here we report that the thiol oxidoreductase PDI is mandatory for activation of the PERK pathway in HEK293T as well as in human pancreatic, lung and colon cancer cells. Under ER stress, depletion of PDI selectively abrogated eIF2α phosphorylation, induction of ATF4, CHOP and even BiP. Furthermore, we could demonstrate that PDI prevented degradation of activated PERK by the 26S proteasome and therefore contributes to maintained PERK signaling. As a result of decreased PERK activity, PDI depleted cells showed an increased vulnerability to ER stress induced by chemicals or ionizing radiation in 2D as well as in 3D culture models. We conclude that PDI is an obligatory regulator of the PERK pathway with future therapy implications.

PDIA3 Reduce Glioma-associated Macrophage/microglia Pro-tumor Activation trough IL-6-STAT3-PDIA3 Pathway.

Background: Glioblastoma (GB - grade IV glioma) is the most aggressive and common cancer of central nervous system with an overall survival of 14-16 months. The GB tumor microenvironment includes cells of the innate immune system identified as glioma-associated microglia/macrophages (GAMs). It is known that between GAMs and GB cells there is a double interaction, but the role of GAMs is still poorly characterized. The endoplasmic reticulum (ER) protein ERp57, also known as PDIA3, is a thiol oxidoreductase with main function related on glycoprotein folding in endoplasmic reticulum. However, PDIA3 shows different functions. In fact, the various subcellular localizations and binding partners of PDIA3 affect numerous physiological processes and diseases: different regulation and modulation of PDIA3 has been reported in multiple pathologies including neurodegenerative diseases and cancer. Methods: In the present work, we evaluated in both GB cells and microglia-macrophage cells the expression of PDIA3 using specimens collected after surgical from 18 GB patients. In addition, we studied in vitro microglia-glioma interaction to determine the role of PDIA3 in viability and the activation of both GB and microglia cells. The study was carried using PDIA3-silenced T98G cells and/or using a pharmacological inhibitor of PDIA3 activity (Punicalagin-PUN).Results: We initially investigated the role of the PDIA3 in GB survival by inquiring The Cancer Genome Atlas dataset. The results indicated that 352 out of 690 patients reported over-expression of PDIA3, which significantly correlated with a ~55% reduction of overall survival. Subsequently, for the first time, we investigated the PDIA3 expression in the tumor and the nearby parenchyma of 18 GB patients and our data showed a significant upregulation (15% vs 10%) of ERp57/PDIA3 in GAMs of tumor specimens respect the microglia present in parenchyma. In addition, we show that conditioned medium (CMs) obtained from both wild type T98G and PDIA3 silenced T98G induced an activation of microglia cells, but the PDIA3 silenced-T98G CMs significant limited the microglia pro-tumor activation probably through a IL-6-STAT3-PDIA3 dependent mechanism. Conclusion: Our data support the relevant role of PDIA3 expression in GB pathology and link the different activation of microglia to a mechanism a IL-6-STAT3-PDIA3 dependent.

Sex-dependent Differences in the Bioenergetics of Liver and Muscle Mitochondria from Mice Containing a Deletion for glutaredoxin-2

Our group recently published a study demonstrating that deleting the gene encoding the matrix thiol oxidoreductase, glutaredoxin-2 (GRX2), alters the bioenergetics of mitochondria isolated from male C57BL/6N mice. Here, we conducted a similar study, examining H2O2 production and respiration in mitochondria isolated from female mice heterozygous (GRX2+/−) or homozygous (GRX2−/−) for glutaredoxin-2. First, we observed that deleting the Grx2 gene does not alter the rate of H2O2 production in liver and muscle mitochondria oxidizing pyruvate, α-ketoglutarate, or succinate. Examination of the rates of H2O2 release from liver mitochondria isolated from male and female mice revealed that (1) sex has an impact on the rate of ROS production by liver and muscle mitochondria and (2) loss of GRX2 only altered ROS release in mitochondria collected from male mice. Assessment of the bioenergetics of these mitochondria revealed that loss of GRX2 increased proton leak-dependent and phosphorylating respiration in liver mitochondria isolated from female mice but did not alter rates of respiration in liver mitochondria from male mice. Furthermore, we found that deleting the Grx2 gene did not alter rates of respiration in muscle mitochondria collected from female mice. This contrasts with male mice where loss of GRX2 substantially augmented proton leaks and ADP-stimulated respiration. Our findings indicate that some fundamental sexual dimorphisms exist between GRX2-deficient male and female rodents.

A universal entropy-driven mechanism for thioredoxin–target recognition

Cysteine residues in cytosolic proteins are maintained in their reduced state, but can undergo oxidation owing to posttranslational modification during redox signaling or under conditions of oxidative stress. In large part, the reduction of oxidized protein cysteines is mediated by a small 12-kDa thiol oxidoreductase, thioredoxin (Trx). Trx provides reducing equivalents for central metabolic enzymes and is implicated in redox regulation of a wide number of target proteins, including transcription factors. Despite its importance in cellular redox homeostasis, the precise mechanism by which Trx recognizes target proteins, especially in the absence of any apparent signature binding sequence or motif, remains unknown. Knowledge of the forces associated with the molecular recognition that governs Trx–protein interactions is fundamental to our understanding of target specificity. To gain insight into Trx–target recognition, we have thermodynamically characterized the noncovalent interactions between Trx and target proteins before S-S reduction using isothermal titration calorimetry (ITC). Our findings indicate that Trx recognizes the oxidized form of its target proteins with exquisite selectivity, compared with their reduced counterparts. Furthermore, we show that recognition is dependent on the conformational restriction inherent to oxidized targets. Significantly, the thermodynamic signatures for multiple Trx targets reveal favorable entropic contributions as the major recognition force dictating these protein–protein interactions. Taken together, our data afford significant new insight into the molecular forces responsible for Trx–target recognition and should aid the design of new strategies for thiol oxidoreductase inhibition.