Phosphoglycerate Kinase Research Articles

Improved Gene Therapy for Metachromatic Leukodystrophy

Metachromatic Leukodystrophy (MLD) is an autosomal recessive lysosomal storage disease (LSD) characterized by a decreased Arylsulfatatse A (ARSA) enzymatic activity. The most common form, late infantile MLD, universally results in rapid loss of neurologic function in early childhood. Ex-vivo hematopoietic stem cell (HSC) gene therapy using a lentiviral vector (LV) can improve clinical outcomes by supplying a functional copy of the ARSA cDNA (Biffi A, et al, Science 2013). Unfortunately, this approach is only successful in pre- and minimally symptomatic individuals and only a small subset of individuals are diagnosed during the limited therapeutic window. As such, the development of additional approaches targeting early symptomatic individuals are critically needed.The only clinical vector (CV) approved to treat MLD patients, PawMut6, includes the human ARSA cDNA gene under the control of the human Phosphoglycerate Kinase (PGK) promoter and includes, in the integrating transcriptional unit, the viral sequences Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) sequence to increase titer and mRNA translation (Biffi A, et al, Science 2013). To increase expression of ARSA cDNA at single integration level, we generated several LVs that include the ARSA gene with a variety of insulators to optimize ARSA expression and enhance safety in transduced cell lines. We placed the ARSA cDNA under the control of the human Elongation Factor 1 alpha (EF1-alpha) promoter, which has been shown to promote higher transcription rates in different cell lines, compared to human PGK as shown by Jane Yuxia Qin, PLos One 2010. Our constructs carry versions of the ARSA gene with and without the 5' and 3' untranslated regions (UTR+ or UTR-) and a Traceable Codon Optimized (TCO) modified sequence to distinguish the transgene from the endogenous ARSA. An ankyrin or foamy insulator have been incorporated to minimize genotoxicity caused by integration events. The WPRE has been proven to enhance the performance of viral vectors. However, to prevent WPRE integration in the host genome, we placed it directly after the 3'-self inactivating LTR (SIN-LTR) together with a strong bovine growth hormone polyA signal (for sequence termination) (BGHpA), as shown by Breda L. et al, Mol Ther 2021.We compared the ARSA activity (normalized to vector copy number (VCN)) of our constructs to that of PawMut6, the LV currently used in clinical trial, on MLD primary patient fibroblast cultures. Our top performing vectors, TCO-EAAWP-UTR +, TCO-EAFWP-UTR - and TCO-AEAFWP-UTR - showed 2X, 10X and 4X more ARSA activity, respectively, compared to that generated using PawMut6. We also detected a superior ability of our vectors to secrete functional ARSA enzyme into the culture media of transduced primary MLD patient fibroblast cells, which is a critical modality for transfer of functional ARSA from microglia to oligodendrocytes. Extracellular vesicle isolation, purification, and immunoblot analysis has demonstrated small vesicle secretion is the primary modality by which ARSA is secreted, having significant implication for how we approach treatment of MLD. In parallel experiments on murine HSC, the TCO-AEAFWP-UTR - vector reproduced similar results, with about 4x more ARSA activity. To exclude potential toxicity, we performed bone marrow transplants on WT animals with HSCs transduced at up to 13 copies per genome. Mice transplanted with high VCN transduced bone marrow did not show signs of bone marrow failure or distress; more extensive evaluation of these animal models is ongoing. Clonogenic assays and secondary transplants are in progress. Upon completion of the in-vivo studies in WT mice, at least two of our best vector candidates will be utilized on a MLD mouse model (ARSA-KO) that we generated using CRISPR-Cas9. Analysis will include pathological sections of the CNS, brain lysate collection and sulfatase activity assays. Our studies are currently focused on completing in-vivo validation and toxicity assays to move our best vector to the pre-clinical and IND application. The accumulated data on our novel vectors imply new mechanistic considerations for treatment of MLD and demonstrate utility as a strong approach for treating early symptomatic patients. DisclosuresVanderver: Homology: Research Funding; Takeda: Research Funding; Ionis and Illumina Inc: Research Funding; Biogen: Research Funding; Eli Lily and Company: Research Funding; Orchard Therapeutics: Research Funding; Gilead Sciences Inc: Research Funding. Adang: MEGMA: Consultancy; Orchard Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Rivella: Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Forma Theraputics: Consultancy; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy.

Rescue of Murine IL-7 Receptor Deficiency with Human IL-7 Receptor Gene Therapy

▪In humans, interleukin 7 (IL-7) receptor (IL-7R) deficiency causes approximately 10% of cases of severe combined immunodeficiency (SCID). IL-7R deficient SCID is a T-B+NK+ SCID and is caused by autosomal recessive deficiency of the IL-7R alpha chain gene (IL7R). IL-7R is a heterodimeric receptor comprised of the alpha chain and the IL-2 receptor common gamma chain (IL2RG). In both mouse and human, IL-7R is a marker of the common lymphoid progenitor cell, and IL-7 signaling leads to STAT5 phosphorylation and proliferation of developing T and B cells. Mice lacking IL7R, Il7r -/-, lack both T and B cells (Peschon, JJ, et al. J Exp Med. 1994). T cells do not progress to TCR beta chain rearrangement and B cell development is halted at the pre-pro-B cell stage. Similar to the mouse, IL-7 signaling in humans is required for T cell receptor beta gene rearrangement and T cell maintenance, however humans lacking IL-7R can develop B cells.A prior attempt to rescue murine IL-7R deficiency utilized a retroviral vector (mouse stem cell virus, MSCV), the MSCV retroviral promoter, and the murine Il7r gene (Jiang, Q, et al. Gene Therapy. 2005). This strategy did restore T cells and had variable restoration of B cells. However, retroviral-based gene addition of Il7r led to a myeloproliferative condition with significant neutrophilia and splenomegaly. Transduced bone marrow cells formed myeloid progenitors in response to IL-7 in vitro. We evaluated a novel gene therapy for IL-7R deficient SCID that utilizes the human IL7R gene. To prevent lineage skewing, we sought to limit ectopic expression of IL7R in non-lymphoid cells by utilizing the endogenous enhancers and promoters of IL7R. These sequences were identified as sites of high sequence conservation across species and DNA accessibility/hypersensitivity (DHS) in human lymphocytes. We are testing these sequences alone or in combination with the constitutive phosphoglycerate kinase promoter (PGK) in VSV-G pseudotyped lentiviral vectors (LV): vPGK_DHS_hIL7R and vDHS_hIL7R. Here we present the first data evaluating the ability of the human IL-7R protein to functionally replace the murine IL-7R protein and the ability of IL7R gene addition to rescue the murine Il7r -/- immunodeficient phenotype in vivo.Transduction of Il7r -/- bone marrow cells with IL7R encoding LV rescued the formation of lymphocyte precursors from murine bone marrow cells in colony forming unit (CFU) assays (pre-B CFU with human IL-7), with the most robust response seen with vPGK_DHS_hIL7R. Mouse bone marrow from Il7r -/- animals transduced ex vivo engrafted in lethally irradiated (8 Gy) Il7r -/-oppositegender recipients and there were no significant aberrations in absolute neutrophil count, hemoglobin or platelet count. Absolute lymphocyte counts in mice receiving transduced Il7r -/-bone marrow cells was higher (mean 2555/μL) than in mice receiving untransduced bone marrow (mean 1410/μL). The proportion of leukocytes that were T cells was 4.2-fold and 9.8-fold higher at 1 and 2 months post-transplant, respectively. B cells were only seen in mice receiving vPGK_DHS_hIL7R: 7.4% of leukocytes versus 1.5% in controls. A reciprocal decrease in the fraction of Gr1+ cells (neutrophils and monocytes) was seen at two months post-transplant in transduced marrow recipients compared to untransduced controls: 36.5% versus 63% Gr1+, respectively. Lymphocyte subsets are being further analyzed, bone marrow and thymic lymphoid precursors assessed, and T and B cell function in response to immunizations are in progress. Further evaluation in human derived IL7R deficient human cells is warranted.For individuals with IL-7R deficient SCID, but no HLA matched hematopoietic stem cell (HSC) donor, there is a difficult choice between the risks of GVHD with a mismatched HSC donor and supportive care with the hope of identifying a matched HSC donor in the future. In immunodeficiencies however age and serious infection are both associated with increased mortality (Pai, SY, et al. NJEM. 2014). This novel approach to IL7R gene replacement has the potential to be a therapeutic and expedient option for those without a matched donor. Additionally, this would be an ideal disorder for HSC conditioning with less toxic, HSC-targeted strategies given gene corrected lymphocytes and progenitors will preferentially expand post-transplant. DisclosuresRivella: Disc Medicine: Consultancy, Membership on an entity's Board of Directors or advisory committees; Keros Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Celgene Corporation: Consultancy; Ionis Pharmaceuticals: Consultancy, Membership on an entity's Board of Directors or advisory committees; MeiraGTx: Consultancy, Membership on an entity's Board of Directors or advisory committees; Forma Theraputics: Consultancy; Incyte: Consultancy.

Lentiviral Gene Therapy for the Correction of Congenital Dyserythropoietic Anemia Type II

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythroid lineage. CDA type II is the most common one: it accounts for around 60% of all cases, and more than 600 cases have been reported so far. CDA II is caused by biallelic mutations in the SEC23B gene and is characterized by ineffective erythropoiesis with morphologic abnormalities of erythroblasts, hemolysis, and secondary iron overload, which is the most frequent complication. Patients usually suffer from variable degrees of jaundice, splenomegaly, and absolute reticulocyte count inadequate depending on the degree of anemia. Hydrops fetalis, aplastic crisis and gallstones are other associated clinical signs. CDA II bone marrow is characterized by the presence of more than 10% mature binucleated erythroblasts. Another distinctive feature of CDA II erythrocytes is hypoglycosylation of membrane proteins. The management of CDA II is generally limited to blood transfusion and iron chelation. Splenectomy has proved to reduce the number of transfusions in CDA II patients. However, allogenic hematopoietic stem cell transplant (HSCT) represents the only curative option for this disease. Autologous HSCT of genetically corrected cells will mean a definitive treatment for CDA II, overcoming the limitations of allogeneic HSCT, such as limited availability of HLA-matched donors, infections linked to immunosuppression or development of graft versus host disease. This strategy has been used to treat many inherited hematological diseases, including red blood cell diseases such as β-thalassemia, sickle cell disease or pyruvate kinase deficiency. Therefore, we have addressed a similar strategy to be applied to CDAII patients.Two different lentiviral vectors carrying either wild type or codon optimized versions of SEC23B cDNA (wtSEC23B LV or coSEC23B LV, respectively) under the control of human phosphoglycerate kinase promoter (PGK) have been developed. Taking advantage of a CDA II model, in which SEC23B knock-out was done in human hematopoietic progenitors through gene editing, we have determined the most effective SEC23B LV version and the most suitable multiplicity of infection (MOI) to compensate protein deficiency. SEC23B knock out human hematopoietic progenitors (CD34 + cells; 80% frame shift mutations; SEC23BKO) showed a sharp reduction in SEC23B protein level. Those SEC23BKO hematopoietic progenitors were transduced with both lentiviral vectors at MOIs ranged from 3 to 25. We observed that SEC23B protein reached physiological or even supraphysiological levels. In addition, the reduction in the number of erythroid colony forming units (CFUs) identified in SEC23BKO CD34 + cells, was partially restored in the LV transduced SEC23BKO progenitors. Significantly, we observed a clear correlation between the used MOI and the vector copy number (VCN) in the CFUs derived from transduced SEC23BKO CD34 + cells. Furthermore, SEC23BKO hematopoietic progenitors were subjected to an in vitro erythroid differentiation protocol. A sharp decrease in the cell growth throughout erythroid differentiation was observed in SEC23BKO condition. However, the transduction with any of SEC23B LVs at MOIs above 10 was able to recover cell expansion to values equal to wild type cells. Interestingly, total level of protein glycosylation during erythroid differentiation was enhanced after SEC23B LV transduction. Glycosylation level in wtSEC23B LV transduced SEC23BKO cells was most similar to the level in wild type cells. Then, we transduced peripheral blood-derived hematopoietic progenitors (PB-CD34 + cells) from a CDA II patient with wtSEC23B LV at MOI 25 and differentiated in vitro to erythroid cells. A complete restauration of SEC23B protein expression and a cell growth increase of wtSEC23B transduced CDAII was observed with vector copy numbers of 0.3 after 14 days under erythroid conditions. More importantly, we could find a decrease in the percentage of bi-/multinucleated erythroid cells generated in vitro after wtSEC23B LV transduction.In summary, SEC23B LV compensate the SEC23B deficiency in SEC23BKO and in CDAII hematopoietic progenitor cells, paving the way for gene therapy of autologous hematopoietic stem and progenitor cell as an alternative and feasible treatment for CDA II. DisclosuresBianchi: Agios pharmaceutics: Consultancy, Membership on an entity's Board of Directors or advisory committees. Sanchez: Bloodgenetics: Other: Co-Founder and promoter; UIC: Current Employment. Ramirez: VIVEBiotech: Current Employment. Segovia: Rocket Pharmaceuticals, Inc.: Consultancy, Research Funding. Quintana Bustamante: Rocket Pharmaceuticals, Inc.: Current equity holder in publicly-traded company.

Impact of Nuclear Factor Erythroid 2-Related Factor 2 in Hepatocellular Carcinoma: Cancer Metabolism and Immune Status.

We examined phosphorylated nuclear factor erythroid 2–related factor 2 (P‐NRF2) expression in surgically resected primary hepatocellular carcinoma (HCC) and investigated the association of P‐NRF2 expression with clinicopathological features and patient outcome. We also evaluated the relationship among NRF2, cancer metabolism, and programmed death ligand 1 (PD‐L1) expression. In this retrospective study, immunohistochemical staining of P‐NRF2 was performed on the samples of 335 patients who underwent hepatic resection for HCC. Tomography/computed tomography using fluorine‐18 fluorodeoxyglucose was performed, and HCC cell lines after NRF2 knockdown were analyzed by array. We also analyzed the expression of PD‐L1 after hypoxia inducible factor 1α (HIF1A) knockdown in NRF2‐overexpressing HCC cell lines. Samples from 121 patients (36.1%) were positive for P‐NRF2. Positive P‐NRF2 expression was significantly associated with high alpha‐fetoprotein (AFP) expression, a high rate of poor differentiation, and microscopic intrahepatic metastasis. In addition, positive P‐NRF2 expression was an independent predictor for recurrence‐free survival and overall survival. NRF2 regulated glucose transporter 1, hexokinase 2, pyruvate kinase isoenzymes L/R, and phosphoglycerate kinase 1 expression and was related to the maximum standardized uptake value. PD‐L1 protein expression levels were increased through hypoxia‐inducible factor 1α after NRF2 overexpression in HCC cells. Conclusions: Our large cohort study revealed that P‐NRF2 expression in cancer cells was associated with clinical outcome in HCC. Additionally, we found that NRF2 was located upstream of cancer metabolism and tumor immunity.

Phosphoglycerate kinase 1 silencing by a novel microRNA microRNA-4523 protects human osteoblasts from dexamethasone through activation of Nrf2 signaling cascade

Nuclear-factor-E2-related factor 2 (Nrf2) cascade activation can ameliorate dexamethasone (DEX)-induced oxidative injury and death in human osteoblasts. Phosphoglycerate kinase 1 (PGK1) depletion is shown to efficiently activate Nrf2 signaling by inducing methylglyoxal modification of Kelch-like ECH-associated protein 1 (Keap1). We here identified a novel PGK1-targeting microRNA: microRNA-4523 (miR-4523). RNA fluorescent in situ hybridization, RNA pull-down, and Argonaute-2 RNA immunoprecipitation results confirmed a direct binding between miR-4523 and PGK1 mRNA in primary human osteoblasts and hFOB1.19 osteoblastic cells. Forced overexpression of miR-4523, using a lentiviral construct, robustly decreased PGK1 3′-UTR (untranslated region) luciferase activity and downregulated its expression in human osteoblasts and hFOB1.19 cells. Furthermore, miR-4523 overexpression activated the Nrf2 signaling cascade, causing Keap1–Nrf2 disassociation, Nrf2 protein stabilization, and its nuclear translocation as well as transcription activation of Nrf2-dependent genes (NQO1, GCLC, and HO1) in human osteoblasts. By expressing a UTR-null PGK1 construct, miR-4523 overexpression-induced Nrf2 cascade activation was however largely inhibited. Importantly, DEX-induced reactive oxygen species production, oxidative injury, and cell apoptosis were significantly attenuated by miR-4523 overexpression in human osteoblasts and hFOB1.19 cells. Such actions by miR-4523 were abolished by Nrf2 shRNA or knockout, but mimicked by PGK1 knockout (using CRISPR/Cas9 method). In PGK1 knockout human osteoblasts, miR-4523 overexpression failed to further increase Nrf2 cascade activation and offer osteoblast cytoprotection against DEX. Significantly, miR-4523 is downregulated in human necrotic femoral head tissues of DEX-taking patients. Together, PGK1 silencing by miR-4523 protected human osteoblasts from DEX through activation of the Nrf2 signaling cascade.

Carbon sources driven supernatant micro-particles differentiate in submerged anaerobic membrane bioreactors (AnMBRs)

Recently, micro-particles with a size range of 0.45–10 μm in anaerobic membrane bioreactors (AnMBRs), were rich in microorganisms and characterized as a significant source to membrane fouling. Nevertheless, little is known about the formation of the micro-particles associated microorganisms as well as their metabolic functions, which could be largely impacted by carbon sources in the influent. Here, protein and glucose were chosen to be two different carbon sources fed into two identical AnMBRs (i.e., AnMBR-P and AnMBR-G), and a comprehensive comparison of the micro-particles formed in corresponding reactors was made. Size-dependent fractionation and fluorescent staining assay showed that micro-particles with the size range of 0.45–1 μm possessed the highest contents (41.4%) in the AnMBR-P and presented as vibrios and coccus, while the 5–10 μm micro-particles with filamentous morphologies were the prevailing fraction (61.9%) in the AnMBR-G. According to the results of high-throughput 16S rRNA gene sequencing, the Lentimicrobiaceae, D8A-2 and Syntrophorhabdus prevailed in the AnMBR-P micro-particles, while the SJA-15 showed overwhelming predominance than the other genera in the AnMBR-G micro-particles. Metagenomic sequencing further revealed that the micro-particles in the AnMBR-P possessed higher abundance in specific functional genes for nitrogen metabolism (nirB), sulfur metabolism (cysK) and cysteine and methionine metabolism (mtaD), as well as in the cell mobility as chemotaxis (rbsB) and flagellar assembly (fliC). In comparison, the micro-particles obtained from the AnMBR-G encoded abundant genes for converting glucose to metabolites in the next level, such as glk (glucokinase), pfk (ATP-dependent phosphofructokinase), and PGK (phosphoglycerate kinase). Overall, our findings provided new insights into the formation of micro-particles in the supernatant of AnMBRs, which could facilitate to propose related fouling alleviation methods from the perspective of carbon sources regulation and/or pretreatment of the influent.

Quantitative Acetylomics Revealed Acetylation-Mediated Molecular Pathway Network Changes in Human Nonfunctional Pituitary Neuroendocrine Tumors.

Acetylation at lysine residue in a protein mediates multiple cellular biological processes, including tumorigenesis. This study aimed to investigate the acetylated protein profile alterations and acetylation-mediated molecular pathway changes in human nonfunctional pituitary neuroendocrine tumors (NF-PitNETs). The anti-acetyl antibody-based label-free quantitative proteomics was used to analyze the acetylomes between NF-PitNETs (n = 4) and control pituitaries (n = 4). A total of 296 acetylated proteins with 517 acetylation sites was identified, and the majority of which were significantly down-acetylated in NF-PitNETs (p<0.05 or only be quantified in NF-PitNETs/controls). These acetylated proteins widely functioned in cellular biological processes and signaling pathways, including metabolism, translation, cell adhesion, and oxidative stress. The randomly selected acetylated phosphoglycerate kinase 1 (PGK1), which is involved in glycolysis and amino acid biosynthesis, was further confirmed with immunoprecipitation and western blot in NF-PitNETs and control pituitaries. Among these acetylated proteins, 15 lysine residues within 14 proteins were down-acetylated and simultaneously up-ubiquitinated in NF-PitNETs to demonstrate a direct competition relationship between acetylation and ubiquitination. Moreover, the potential effect of protein acetylation alterations on NF-PitNETs invasiveness was investigated. Overlapping analysis between acetylomics data in NF-PitNETs and transcriptomics data in invasive NF-PitNETs identified 26 overlapped molecules. These overlapped molecules were mainly involved in metabolism-associated pathways, which means that acetylation-mediated metabolic reprogramming might be the molecular mechanism to affect NF-PitNET invasiveness. This study provided the first acetylomic profiling and acetylation-mediated molecular pathways in human NF-PitNETs, and offered new clues to elucidate the biological functions of protein acetylation in NF-PitNETs and discover novel biomarkers for early diagnosis and targeted therapy of NF-PitNETs.

Stabilization and Kinetics of an Adsorbed Protein Depends on the Poly(N-isopropylacrylamide) Grafting Density.

The solubility transition at the lower critical solution temperature (LCST, 32 °C) of poly(N-isopropylacrylamide) (PNIPAM) is widely used as a thermal switch to rapidly and reversibly capture and release proteins and cells. It is generally assumed that proteins adsorbed to PNIPAM above the LCST are unaffected by polymer interactions. Here we show that the folding stability of the enzyme phosphoglycerate kinase (PGK) is increased by interactions with end-grafted PNIPAM films above the LCST. We systematically compare two protein mutants with different stabilities. The stabilization mirrors the degree of protein adsorption under grafting conditions studied previously. Maximum stabilization occurs when proteins adsorb to low density, collapsed polymer "mushrooms". In the denser polymer "brush" regime, protein stabilization decreases back to a value indistinguishable from the bulk solution, consistent with low protein adsorption on dense, collapsed brushes. The temperature-dependent kinetics measured by Fast Relaxation Imaging reveals that PNIPAM does not affect the overall folding/unfolding mechanism. Based on the different stabilizations of two mutants and the relaxation kinetics, we hypothesize that the polymer acts mainly by increasing the conformational entropy of the folded protein by interacting with the protein surface and less by crowding the unfolded state of PGK.

Impaired age-dependent increases in phosphoglycerate kinase activity in red blood cells of Parkinson's disease patients

BackgroundImpaired bioenergetics are partially involved in the pathogenesis of Parkinson's disease (PD). Phosphoglycerate kinase (PGK), an essential enzyme for glycolysis, has recently attracted attention due to its pathogenic role in PD and as a target for disease-modifying therapies. This study is aimed to evaluate the profiles of PGK activity in red blood cells (RBCs) of PD patients and controls. MethodsSixty-eight PD patients and thirty-four age-matched unrelated controls were enrolled. PGK activities of RBCs were measured by the established colorimetric assay and standardized by the same RBC samples. ResultsPGK activity of the PD group was significantly higher than that of the control group in participants aged sixty-five years or younger, whereas it was not significantly different between the two groups at any age. PGK activity was positively correlated with aging in the control group, but this was not noted in the PD group. On multivariable analysis by partial correlation in the PD group, PGK activity was negatively correlated with the specific binding ratio of dopamine transporter scintigraphy in the striatum. The levodopa-equivalent daily dose was not significantly correlated with the enzyme activity. ConclusionThe results support the following: 1) elevation of PGK activities in RBCs can be detected in relatively young PD patients and with normal aging; 2) the degree of striatonigral degeneration is associated with elevated PGK activities. These are important considerations when the PGK assay is applied as a diagnostic biomarker of PD and to therapeutically monitor PGK-enhancing treatments.

Exploring the Bile Stress Response of Lactobacillus mucosae LM1 through Exoproteome Analysis.

Lactobacillus sp. have long been studied for their great potential in probiotic applications. Recently, proteomics analysis has become a useful tool for studies on potential lactobacilli probiotics. Specifically, proteomics has helped determine and describe the physiological changes that lactic acid bacteria undergo in specific conditions, especially in the host gut. In particular, the extracellular proteome, or exoproteome, of lactobacilli contains proteins specific to host– or environment–microbe interactions. Using gel-free, label-free ultra-high performance liquid chromatography tandem mass spectrometry, we explored the exoproteome of the probiotic candidate Lactobacillus mucosae LM1 subjected to bile treatment, to determine the proteins it may use against bile stress in the gut. Bile stress increased the size of the LM1 exoproteome, secreting ribosomal proteins (50S ribosomal protein L27 and L16) and metabolic proteins (lactate dehydrogenase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate dehydrogenases, among others) that might have moonlighting functions in the LM1 bile stress response. Interestingly, membrane-associated proteins (transporters, peptidase, ligase and cell division protein ftsH) were among the key proteins whose secretion were induced by the LM1 bile stress response. These specific proteins from LM1 exoproteome will be useful in observing the proposed bile response mechanisms via in vitro experiments. Our data also reveal the possible beneficial effects of LM1 to the host gut.

A combination strategy of solubility enhancers for effective production of soluble and bioactive human enterokinase

Enterokinase is one of the hydrolases that catalyze hydrolysis to regulate biological processes in intestinal visceral mucosa. Enterokinase plays an essential role in accelerating the process of protein digestion as it converts trypsinogen into active trypsin by accurately recognizing and cleaving a specific peptide sequence, (Asp)4-Lys. Due to its exceptional substrate specificity, enterokinase is widely used as a versatile molecular tool in various bioprocessing, especially in removing fusion tags from recombinant proteins. Despite its biotechnological importance, mass production of soluble enterokinase in bacteria still remains an unsolved challenge. Here, we present an effective production strategy of human enterokinase using tandemly linked solubility enhancers consisting of thioredoxin, phosphoglycerate kinase or maltose-binding protein. The resulting enterokinases exhibited significantly enhanced solubility and bacterial expression level while retaining enzymatic activity, which demonstrates that combinatorial design of fusion proteins has the potential to provide an efficient way to produce recombinant proteins in bacteria.

PGK1 : An Essential Player in Modulating Tumor Metabolism.

Phosphoglycerate kinase 1 (PGK1) is the first enzyme in glycolysis to generate a molecule of ATP in the conversion of 1,3-bisphosphoglycerate (1,3-BPG) to 3-phosphoglycerate (3-PG). In addition to the role of glycolysis, PGK-1 acts as a polymerase alpha cofactor protein, with effects on the tricarboxylic acid cycle, DNA replication and repair. Posttranslational modifications such as methylation, phosphorylation, and acetylation have been seen to activate PGK1 in cancer. High levels of intracellular PGK1 are associated with tumorigenesis and progression, and chemoradiotherapy resistance. However, high levels of extracellular PGK1 suppress angiogenesis and subsequently counteract cancer malignancy. Here we have summarized the current knowledge on the mechanisms and effects of PGK1 in various tumor types and evaluated its potential prognostic and therapeutic value in cancer. The data summarized here aims at providing molecular information and new ideas of employing natural products to combat cancer associated with PGK1.

Proteomic Analysis Reveals Growth Inhibition of Coriolus versicolor by Methanol Extracts of Cinnamomum camphora Xylem

The extracts of decay-resistant tree species are important research objects for the future development of wood preservatives. To understand the antifungal mechanisms of Coriolus versicolor inhibition with methanol extracts of C. camphora xylem, the protein profiles of C. versicolor were analyzed using 2-DE followed by MALDI-TOF/MS and bioinformatic analyses. The results showed that 41 protein spots were obviously changed among the 366-385 protein spots of C. versicolor treated with methanol extracts of C. camphora xylem. Twenty-one protein spots were upregulated, and 20 protein spots were downregulated. Cellular localization was performed to identify these differential proteins, and biological process and functional analysis found that 9 of these proteins were in the cytoplasm, 6 were intracellular, and 5 were in the mitochondrion. A total of 18.8% were mapped to small-molecule metabolic processes, 12.5% to cellular amino acid metabolic processes, and 10.9% to cellular nitrogen compound metabolic processes. Twenty-five percent of the differential proteins were associated with ion bonding, 15% with oxidoreductase activity, and 15% with ATPase activity and transmembrane transport activity. Downregulated expression of aspartate aminotransferase, ATP synthase alpha chain, DEAD/DEAH-box helicase, and phosphoglycerate kinase showed that the methanol extracts of C. camphora xylem disrupted functional aspects such as nitrogen and carbon metabolism, energy metabolism, hormone signal response, and glucose metabolism, eventually leading to C. versicolor inhibition.

Thylakoid proteome variation of Eutrema salsugineum in response to drought and salinity combined stress

It is well known that plant responses to stress involve different events occurring at different places of the cell/leaf and at different time scales in relation with the plant development. In fact, the organelles proteomes include a wide range of proteins that could include a wide range of proteins showing a considerable change in cellular functions and metabolism process. On this basis, a comparative proteomics analysis and fluorescence induction measurements were performed to investigate the photosynthetic performance and the relative thylakoid proteome variation in Eutrema salsugineum cultivated under salt stress (200 mM NaCl), water deficit stress (PEG) and combined treatment (PEG + NaCl) as a hyperosmotic stress. The obtained results showed a significant decrease of plant growth under drought stress conditions, with the appearance of some toxicity symptoms, especially in plants subjected to combined treatment. Application of salt or water stress alone showed no apparent change in the chlorophyll a fluorescence transients, primary photochemistry (fluorescence kinetics of the O-J phase), the PQ pool state (J-I phase changes), (Fv/Fm) and (Fk/Fj) ratios. However, a considerable decrease of all these parameters was observed under severe osmotic stress (PEG + NaCl). The thylakoid proteome analysis revealed 58 proteins showing a significant variation in their abundance between treatments (up or down regulation). The combined treatment (PEG + NaCl) induced a decrease in the expression of the whole PSII core subunit (D1, D2, CP43, CP47, PsbE and PsbH), whereas the OEC subunits proteins remained constant. An increase in the amount of PsaD, PsaE, PsaF, PsaH, PsaK and PsaN was detected under drought stress (PEG5%). No significant change in the accumulation of Cyt b6 and Cyt f was observed. Some regulated proteins involved in cellular redox homeostasis were detected (glutamine synthetase, phosphoglycerate kinase, transketolase), and showed a significant decrease under the combined treatment. Some oxidative stress related proteins were significantly up-regulated under salt or drought stress and could play a crucial role in the PSI photoprotection and the control of ROS production level.

PGK1-coupled HSP90 stabilizes GSK3β expression to regulate the stemness of breast cancer stem cells.

Objective:Glycogen synthase kinase-3β (GSK3β) has been recognized as a suppressor of Wnt/β-catenin signaling, which is critical for the stemness maintenance of breast cancer stem cells. However, the regulatory mechanisms of GSK3β protein expression remain elusive.Methods:Co-immunoprecipitation and mass spectral assays were performed to identify molecules binding to GSK3β, and to characterize the interactions of GSK3β, heat shock protein 90 (Hsp90), and co-chaperones. The role of PGK1 in Hsp90 chaperoning GSK3β was evaluated by constructing 293T cells stably expressing different domains/mutants of Hsp90α, and by performing a series of binding assays with bacterially purified proteins and clinical specimens. The influences of Hsp90 inhibitors on breast cancer stem cell stemness were investigated by Western blot and mammosphere formation assays.Results:We showed that GSK3β was a client protein of Hsp90. Hsp90, which did not directly bind to GSK3β, interacted with phosphoglycerate kinase 1 via its C-terminal domain, thereby facilitating the binding of GSK3β to Hsp90. GSK3β-bound PGK1 interacted with Hsp90 in the “closed” conformation and stabilized GSK3β expression in an Hsp90 activity-dependent manner. The Hsp90 inhibitor, 17-AAG, rather than HDN-1, disrupted the interaction between Hsp90 and PGK1, and reduced GSK3β expression, resulting in significantly reduced inhibition of β-catenin expression, to maintain the stemness of breast cancer stem cells.Conclusions:Our findings identified a novel regulatory mechanism of GSK3β expression involving metabolic enzyme PGK1-coupled Hsp90, and highlighted the potential for more effective cancer treatment by selecting Hsp90 inhibitors that do not affect PGK1-regulated GSK3β expression.

Pathological Role of Phosphoglycerate Kinase 1 in Balloon Angioplasty-Induced Neointima Formation

Restenosis is a common vascular complication after balloon angioplasty. Catheter balloon inflation-induced transient ischemia (hypoxia) of local arterial tissues plays a pathological role in neointima formation. Phosphoglycerate kinase 1 (PGK1), an adenosine triphosphate (ATP)-generating glycolytic enzyme, has been reported to associate with cell survival and can be triggered under hypoxia. The purposes of this study were to investigate the possible role and regulation of PGK1 in vascular smooth muscle cells (VSMCs) and balloon-injured arteries under hypoxia. Neointimal hyperplasia was induced by a rat carotid artery injury model. The cellular functions and regulatory mechanisms of PGK1 in VSMCs were investigated using small interfering RNAs (siRNAs), chemical inhibitors, or anaerobic cultivation. Our data indicated that protein expression of PGK1 can be rapidly induced at a very early stage after balloon angioplasty, and the silencing PGK1-induced low cellular energy circumstance resulted in the suppressions of VSMC proliferation and migration. Moreover, the experimental results demonstrated that blockage of PDGF receptor-β (PDGFRB) or its downstream pathway, the phosphoinositide 3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) axis, effectively reduced hypoxia-induced factor-1 (HIF-1α) and PGK1 expressions in VSMCs. In vivo study evidenced that PGK1 knockdown significantly reduced neointima hyperplasia. PGK1 was expressed at the early stage of neointimal formation, and suppressing PGK1 has a potential beneficial effect for preventing restenosis.

P–026 Seminal plasma exosomes: a promising source of biomarkers for fertility evaluation

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

Exposure to Phosphoglycerate Kinase 1 Activators and Incidence of Parkinson's Disease.

Preclinical and epidemiological data suggest that phosphoglycerate kinase 1 activators could have neuroprotective properties and prevent PD. The objective of this study was to compare the association between increased use of phosphoglycerate kinase 1 activators and increased use of tamsulosin with PD incidence. Our retrospective cohort study included men older than age 66 years newly exposed to phosphoglycerate kinase 1 activators or tamsulosin and compared their PD incidence, using health care administrative data of Ontario, Canada. Among 265,745 men, each additional year of cumulative use of phosphoglycerate kinase 1 activators or tamsulosin was associated with 6% and 8% reduction, respectively, in the hazard of PD incidence. These hazards were not significantly different (P= 0.2094). A secondary analysis with the observation window starting after 6 months and 1 and 2 years showed similar results. Increasing exposure to phosphoglycerate kinase 1 activators and tamsulosin were both associated with small reductions in PD incidence. These results support further investigation of phosphoglycerate kinase 1 activators and tamsulosin for possible PD disease-modifying properties. © 2021 International Parkinson and Movement Disorder Society.

A nitrogen stress-inducible small RNA regulates CO2 fixation in Nostoc.

In the absence of fixed nitrogen, some filamentous cyanobacteria differentiate heterocysts, specialized cells devoted to fixing atmospheric nitrogen (N2). This differentiation process is controlled by the global nitrogen regulator NtcA and involves extensive metabolic reprogramming, including shutdown of photosynthetic CO2 fixation in heterocysts, to provide a microaerobic environment suitable for N2 fixation. Small regulatory RNAs (sRNAs) are major post-transcriptional regulators of gene expression in bacteria. In cyanobacteria, responding to nitrogen deficiency involves transcribing several nitrogen-regulated sRNAs. Here, we describe the participation of nitrogen stress-inducible RNA 4 (NsiR4) in post-transcriptionally regulating the expression of two genes involved in CO2 fixation via the Calvin cycle: glpX, which encodes bifunctional sedoheptulose-1,7-bisphosphatase/fructose-1,6-bisphosphatase (SBPase), and pgk, which encodes phosphoglycerate kinase (PGK). Using a heterologous reporter assay in Escherichia coli, we show that NsiR4 interacts with the 5′-untranslated region (5′-UTR) of glpX and pgk mRNAs. Overexpressing NsiR4 in Nostoc sp. PCC 7120 resulted in a reduced amount of SBPase protein and reduced PGK activity, as well as reduced levels of both glpX and pgk mRNAs, further supporting that NsiR4 negatively regulates these two enzymes. In addition, using a gfp fusion to the nsiR4 promoter, we show stronger expression of NsiR4 in heterocysts than in vegetative cells, which could contribute to the heterocyst-specific shutdown of Calvin cycle flux. Post-transcriptional regulation of two Calvin cycle enzymes by NsiR4, a nitrogen-regulated sRNA, represents an additional link between nitrogen control and CO2 assimilation.

Abstract 1974: C3G down-regulation in glioblastoma induces a pro-invasive and glycolytic phenotype, accompanied by RTKs dysregulation

Abstract C3G is a guanine-nucleotide exchange factor (GEF) for Rap1, although it can act through GEF-independent mechanisms. C3G plays a dual role in cancer, acting as either a tumor suppressor or inducer depending on the tumor type/stage. It regulates different key aspects of the tumorigenic process such as invasion, apoptosis or proliferation. In colon carcinoma, C3G represses invasion through down-regulation of p38αMAPK activity and promotes tumor growth. We have now analyzed the role of C3G in glioblastoma (GBM), a tumor characterized by its aggressiveness and disseminative capacity. To do it, we have used different experimental approaches: permanent gene silencing, knock-out using CRISPR/Cas9 technology and transient overexpression in U87 cell line and patient-derived GBM cells. We found that C3G down-regulation enhances invasion through the induction of an epithelial/glial to mesenchymal transition-like process. C3G deficiency also facilitates foci generation in anchorage-dependent and independent growth assays, but with lower cell density as a consequence of a reduced proliferation. In in vivo analyses, cells with C3G knock-down generated larger tumors in xenografts and chick chorioallantoic membrane xenografts assays with increased angiogenesis and α-SMA+ fibroblasts, but a lower proliferation. Mechanistically, C3G down-regulation impairs EGF/EGFR signaling by decreasing EGFR cell membrane localization, leading to a reduction in EGF/EGFR-induced invasiveness. In contrast, C3G down-regulation promotes the activation of several receptor tyrosine kinases (RTKs) that might promote invasion. In particular, the enhanced FGF2/FGFR1 signaling increases invasion in C3G-silenced cells, through a mechanism likely dependent on ERKs. Moreover, a proteomic analysis revealed that C3G down-regulation increases the levels of different key glycolytic enzymes, such as aldolase, phosphoglycerate kinase, enolase and pyruvate kinase (PK). The activity of PK and lactate dehydrogenase as well as lactate release to the extracellular environment were also increased upon C3G silencing. In conclusion, our data demonstrate that C3G inhibits invasion of GBM cells, while increasing their proliferative capacity. Moreover, we show a distinct dependency on C3G for EGF/EGFR signaling versus other RTKs, suggesting that assessing C3G levels may discriminate GBM patient responders to different RTK inhibitors. In addition, our results indicate that C3G regulates not only GBM growth and invasiveness, but it also contributes to reprogram its glycolytic metabolism. Hence, low levels of C3G correlate with a more mesenchymal and glycolytic phenotype with enhanced aggressiveness and worse patient prognosis. This promising role of C3G as a key player in GBM should be further characterized to define its prognostic value and its potential relevance as a predictor of the response to therapy. Citation Format: Sara Manzano, Óscar Herranz, Paloma Bragado, Patricia Jáuregui, María Rodrigo, Celia Sequera, Cristina Baquero, Nerea Palao, Ignacio Rubio, Álvaro Gutierrez-Uzquiza, Carmen Guerrero, Almudena Porras. C3G down-regulation in glioblastoma induces a pro-invasive and glycolytic phenotype, accompanied by RTKs dysregulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1974.