SUMMARY N-acetyl-aspartyl-glutamate (NAAG) is a peptide-based neurotransmitter that has been extensively studied in many neurological diseases. In this study, we show a specific role of NAAG in cancer. We found that NAAG is more abundant in higher grade cancers and is a source of glutamate in cancers expressing glutamate carboxypeptidase II (GCPII), the enzyme that hydrolyzes NAAG to glutamate and N-acetyl-aspartate (NAA). Knocking down GCPII expression through genetic alteration or pharmacological inhibition of GCPII results in a reduction of both glutamate concentrations and cancer growth. Moreover, targeting GCPII in combination with glutaminase inhibition accentuates these effects. These findings suggest that NAAG serves as an important reservoir to provide glutamate to cancer cells through GCPII when glutamate production from other sources is limited. Thus, GCPII is a viable target for cancer therapy, either alone or in combination with glutaminase inhibition.
One of the key quality attributes of monoclonal antibodies is the glycan pattern and distribution. Two terminal galactose residues typically represent a small fraction of the total glycans from antibodies. However, antibodies with defined glycosylation properties including enhanced galactosylation have been shown to exhibit altered properties for these important biomedical modalities. In this study, the disruption of two α-2,3 sialyltransferases (ST3GAL4 and ST3GAL6) from Chinese Hamster Ovary (CHO) cells was combined with protein engineering of the Fc region to generate an IgG containing 80% bigalactosylated and fucosylated (G2F) glycoforms. Expression of the same single amino acid mutant (F241A) IgG in CHO cells with a triple gene knockout of fucosyltransferase (FUT8) plus ST3GAL4 and ST3GAL6 lowered the galactosylation glycoprofile to 65% bigalactosylated G2 glycans. However, overexpression of IgGs with four amino acid substitutions recovered the G2 glycoform composition approximately 80%. Combining genome and protein engineering in CHO cells will provide a new antibody production platform that enables biotechnologists to generate glycoforms standards for specific biomedical and biotechnology applications.
Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.
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Chinese hamster ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13 C-glucose, 13 C-glutamine, and 13 C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition.Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-acetyl-glucosamine and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13 C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.
Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of lymphoid neoplasms with different biological characteristics. About 90% of all lymphomas in the United States originate from B lymphocytes, while the remaining originate from T cells [1]. The treatment of NHLs depends on the neoplastic histology and stage of the tumor, which will indicate whether radiotherapy, chemotherapy, or a combination is the best suitable treatment [2]. The American Cancer Society describes the staging of lymphoma as follows: Stage I is lymphoma in a single node or area. Stage II is when that lymphoma has spread to another node or organ tissue. Stage III is when it has spread to lymph nodes on two sides of the diaphragm. Stage IV is when cancer has significantly spread to organs outside the lymph system. Radiation therapy is the traditional therapeutic route for localized follicular and mucosa-associated lymphomas. Chemotherapy is utilized for the treatment of large-cell lymphomas and high-grade lymphomas [2]. However, the treatment of indolent lymphomas remains problematic as the patients often have metastasis, for which no standard approach exists [2].
Non-Hodgkin lymphomas (NHLs) are a heterogeneous group of lymphoid neoplasms with differing biological characteristics. About 90% of all lymphomas in the United States originate from B lymphocytes, while the remaining originate from T cells [1]. The treatment of NHLs depends on neoplastic histology and the stage of the tumor, which will indicate whether radiotherapy, chemotherapy, or a combination is the best suitable treatment [2]. The American Cancer Society describes the staging of lymphoma as follows: Stage I is lymphoma in a single node or area. Stage II is when that lymphoma has spread to another node or organ tissue. Stage III is when it has spread to lymph nodes in two sides of the diaphragm. Stage IV is when the cancer has significantly spread to organs outside the lymph system. Radiation therapy is the traditional therapeutic route for localized follicular and mucosa-associated lymphomas. Chemotherapy is utilized for the treatment of large cell lymphomas and high-grade lymphomas [2]. However, treatment of indolent lymphomas remains problematic as the patients often have metastasis for which no standard approach exists [2].
Barth syndrome (BTHS) is an X-linked genetic condition caused by defects in TAZ, which encodes a transacylase involved in the remodeling of the inner mitochondrial membrane phospholipid, cardiolipin (CL). As such, CL has been implicated in numerous mitochondrial functions, and the role of defective CL in the clinical pathology of BTHS is under intense investigation. We used untargeted proteomics, shotgun lipidomics, gene expression analysis, and targeted metabolomics to identify novel areas of mitochondrial dysfunction in a new model of TAZ deficiency in HEK293 cells. Functional annotation analysis of proteomics data revealed abnormal regulation of mitochondrial respiratory chain complex I (CI), driven by the reduced abundance of 6 CI associated proteins in TAZ-deficient HEK293 cells: MT-ND3, NDUFA5, NDUFAB1, NDUFB2, NDUFB4, and NDUFAF1. This resulted in reduced assembly and function of CI in TAZ-deficient HEK293 cells as well as BTHS patient derived lymphoblast cells. We also identified increased abundance of PARL, a rhomboid protein involved in the regulation of mitophagy and apoptosis, and abnormal downstream processing of PGAM5, another mediator of mitochondrial quality control, in TAZ-deficient cells. Lastly, we modulated CL via the phospholipase inhibitor bromoenol lactone and the CL targeted SS-peptide, SS-31, and showed that each is able to remediate abnormalities in CI abundance as well as PGAM5 processing. Thus, mitochondrial respiratory chain CI and PARL/PGAM5 regulated mitochondrial quality control, both of whose functions localize to the inner mitochondrial membrane, are dysregulated due to TAZ deficiency and are partially remediated via modulation of CL.
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