Dataset Information

The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo.

ABSTRACT: We here examined the potential biological function of phosphoenolpyruvate carboxykinase 1 (PCK1) in angiogenesis. shRNA- or CRISPR-Cas9-induced PCK1 depletion potently inhibited endothelial cell proliferation, migration, sprouting, and tube formation, whereas ectopic PCK1 overexpression exerted opposite activity. In HUVECs, Gα_i3 expression and Akt activation were decreased following PCK1 depletion, but were augmented by ectopic PCK1 overexpression. In vivo, retinal expression of PCK1 gradually increased from postnatal day 1 (P1) to P5. The intravitreous injection of endothelial-specific PCK1 shRNA adenovirus at P1 potently inhibited the radial extension of vascular plexus at P5. Conditional endothelial knockdown of PCK1 in adult mouse retina increased vascular leakage and the number of acellular capillaries while decreasing the number of RGCs in murine retinas. In diabetic retinopathy patients, PCK1 mRNA and protein levels were up-regulated in retinal tissues. Together, PCK1 is essential for angiogenesis possibly by mediating Gα_i3 expression and Akt activation.

SUBMITTER: Yao J

PROVIDER: S-EPMC9140980 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo.

Yao Jin J Wu Xin-Yuan XY Yu Qing Q Yang Shuo-Fei SF Yuan Jin J Zhang Zhi-Qing ZQ Xue Jin-Song JS Jiang Qin Q Chen Min-Bin MB Xue Guan-Hua GH Cao Cong C

Science advances 20220527 21

We here examined the potential biological function of phosphoenolpyruvate carboxykinase 1 (PCK1) in angiogenesis. shRNA- or CRISPR-Cas9-induced PCK1 depletion potently inhibited endothelial cell proliferation, migration, sprouting, and tube formation, whereas ectopic PCK1 overexpression exerted opposite activity. In HUVECs, Gα<sub>i3</sub> expression and Akt activation were decreased following PCK1 depletion, but were augmented by ectopic PCK1 overexpression. In vivo, retinal expression of PCK1 ...[more]

PMID: 35622925

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Project description:Tuberculosis, the second leading infectious disease killer after HIV, remains a top public health priority. The causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), which can cause both acute and clinically latent infections, reprograms metabolism in response to the host niche. Phosphoenolpyruvate carboxykinase (Pck) is the enzyme at the center of the phosphoenolpyruvate-pyruvate-oxaloacetate node, which is involved in regulating the carbon flow distribution to catabolism, anabolism, or respiration in different states of Mtb infection. Under standard growth conditions, Mtb Pck is associated with gluconeogenesis and catalyzes the metal-dependent formation of phosphoenolpyruvate. In non-replicating Mtb, Pck can catalyze anaplerotic biosynthesis of oxaloacetate. Here, we present insights into the regulation of Mtb Pck activity by divalent cations. Through analysis of the X-ray structure of Pck-GDP and Pck-GDP-Mn2+ complexes, mutational analysis of the GDP binding site, and quantum mechanical (QM)-based analysis, we explored the structural determinants of efficient Mtb Pck catalysis. We demonstrate that Mtb Pck requires presence of Mn2+ and Mg2+ cations for efficient catalysis of gluconeogenic and anaplerotic reactions. The anaplerotic reaction, which preferably functions in reducing conditions that are characteristic for slowed or stopped Mtb replication, is also effectively activated by Fe2+ in the presence of Mn2+ or Mg2+ cations. In contrast, simultaneous presence of Fe2+ and Mn2+ or Mg2+ inhibits the gluconeogenic reaction. These results suggest that inorganic ions can contribute to regulation of central carbon metabolism by influencing the activity of Pck. Furthermore, the X-ray structure determination, biochemical characterization, and QM analysis of Pck mutants confirmed the important role of the Phe triad for proper binding of the GDP-Mn2+ complex in the nucleotide binding site and efficient catalysis of the anaplerotic reaction.

Dataset Information

The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo.

Publications

The requirement of phosphoenolpyruvate carboxykinase 1 for angiogenesis in vitro and in vivo.

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