A Small Region in the Angiotensin-Converting Enzyme Distal Ectodomain Is Required for Cleavage-Secretion of the Protein at the Plasma Membrane

Chattopadhyay, Saurabh; Karan, Goutam; Sen, Indira; Sen, Ganes C.

doi:10.1021/bi800702a

Cited by 4 publications

(4 citation statements)

References 52 publications

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“…) were expressed in mammalian cells and then purified using lisinopril‐sepharose affinity chromatography. The sACE, C‐sACE and N‐sACE constructs are expressed as membrane‐bound proteins and undergo poor ectodomain shedding . Hence, these constructs were purified directly from cell lysate.…”

Section: Resultsmentioning

confidence: 99%

Kinetic and structural characterization of amyloid‐β peptide hydrolysis by human angiotensin‐1‐converting enzyme

et al. 2016

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Angiotensin‐1‐converting enzyme (ACE), a zinc metallopeptidase, consists of two homologous catalytic domains (N and C) with different substrate specificities. Here we report kinetic parameters of five different forms of human ACE with various amyloid beta (Aβ) substrates together with high resolution crystal structures of the N‐domain in complex with Aβ fragments. For the physiological Aβ(1–16) peptide, a novel ACE cleavage site was found at His14‐Gln15. Furthermore, Aβ(1–16) was preferentially cleaved by the individual N‐domain; however, the presence of an inactive C‐domain in full‐length somatic ACE (sACE) greatly reduced enzyme activity and affected apparent selectivity. Two fluorogenic substrates, Aβ(4–10)Q and Aβ(4–10)Y, underwent endoproteolytic cleavage at the Asp7‐Ser8 bond with all ACE constructs showing greater catalytic efficiency for Aβ(4–10)Y. Surprisingly, in contrast to Aβ(1–16) and Aβ(4–10)Q, sACE showed positive domain cooperativity and the double C‐domain (CC‐sACE) construct no cooperativity towards Aβ(4–10)Y. The structures of the Aβ peptide–ACE complexes revealed a common mode of peptide binding for both domains which principally targets the C‐terminal P2′ position to the S2′ pocket and recognizes the main chain of the P1′ peptide. It is likely that N‐domain selectivity for the amyloid peptide is conferred through the N‐domain specific S2′ residue Thr358. Additionally, the N‐domain can accommodate larger substrates through movement of the N‐terminal helices, as suggested by the disorder of the hinge region in the crystal structures. Our findings are important for the design of domain selective inhibitors as the differences in domain selectivity are more pronounced with the truncated domains compared to the more physiological full‐length forms.DatabaseThe atomic coordinates and structure factors for N‐domain ACE with Aβ peptides 4–10 (5AM8), 10–16 (5AM9), 1–16 (5AMA), 35–42 (5AMB) and (4–10)Y (5AMC) complexes have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org/).

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Section: Resultsmentioning

confidence: 99%

Kinetic and structural characterization of amyloid‐β peptide hydrolysis by human angiotensin‐1‐converting enzyme

et al. 2016

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“…However, ACE mRNA levels in isolated W/W v cardiomyocytes were similar to those in WT cardiomyocytes (0.066 ± 0.009 versus 0.0512 ± 0.003 ACE/GAPDH mRNA transcript ratio in WT and W/W v , respectively; n = 6-7 cardiomyocyte isolations/group). It is tempting to speculate that decreased shedding of cell surface ACE, which may be regulated by ACE secretase (20), rather than increased transcription, produced higher ACE levels in W/W v LVs; however, this increase is unlikely to be due to suppressed chymase-dependent Ang II formation because, in WT mice, chronic inhibition of mouse MC protease-4 (MMCP4), the major Ang II-forming chymase in the LV (see below), did not increase LV ACE immunoreactivity (data not shown).…”

Section: Mcs Are the Major Source Of The Non-ace Ang Ii-forming Activmentioning

confidence: 99%

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

Wei¹,

Hase²,

Inoue³

et al. 2010

J. Clin. Invest.

131

147

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Ang I-converting enzyme (ACE) inhibitors are widely believed to suppress the deleterious cardiac effects of Ang II by inhibiting locally generated Ang II. However, the recent demonstration that chymase, an Ang IIforming enzyme stored in mast cell granules, is present in the heart has added uncertainty to this view. As discussed here, using microdialysis probes tethered to the heart of conscious mice, we have shown that chronic ACE inhibitor treatment did not suppress Ang II levels in the LV interstitial fluid (ISF) despite marked inhibition of ACE. However, chronic ACE inhibition caused a marked bradykinin/B2 receptor-mediated increase in LV ISF chymase activity that was not observed in mast cell-deficient Kit W /Kit W-v mice. In chronic ACE inhibitor-treated mast cell-sufficient littermates, chymase inhibition decreased LV ISF Ang II levels substantially, indicating the importance of mast cell chymase in regulating cardiac Ang II levels. Chymase-dependent processing of other regulatory peptides also promotes inflammation and tissue remodeling. We found that combined chymase and ACE inhibition, relative to ACE inhibition alone, improved LV function, decreased adverse cardiac remodeling, and improved survival after myocardial infarction in hamsters. These results suggest that chymase inhibitors could be a useful addition to ACE inhibitor therapy in the treatment of heart failure.

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“…The ectodomain of ACE can be cleaved by a membrane-bound metalloprotease, to generate a soluble form [16]–[18]. Although ACE secretase is unknown, its activity has been characterized as a membrane-associated protease.…”

Section: Introductionmentioning

confidence: 99%

“…ACE is expressed on the cell surface as type I ectoprotein, with a long ectodomain containing the enzymatic active site, short cytoplasmic domain and a transmembrane domain [13] – [15] . The ectodomain of ACE can be cleaved by a membrane-bound metalloprotease, to generate a soluble form [16] – [18] . Although ACE secretase is unknown, its activity has been characterized as a membrane-associated protease.…”

Section: Introductionmentioning

confidence: 99%

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

et al. 2014

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Angiotensin-converting enzyme (ACE) regulates normal blood pressure and fluid homeostasis through its action in the renin-angiotensin-system (RAS). Ace-/- mice are smaller in size, have low blood pressure and defective kidney structure and functions. All of these defects are cured by transgenic expression of somatic ACE (sACE) in vascular endothelial cells of Ace-/- mice. sACE is expressed on the surface of vascular endothelial cells and undergoes a natural cleavage secretion process to generate a soluble form in the body fluids. Both the tissue-bound and the soluble forms of ACE are enzymatically active, and generate the vasoactive octapeptide Angiotensin II (Ang II) with equal efficiency. To assess the relative physiological roles of the secreted and the cell-bound forms of ACE, we expressed, in the vascular endothelial cells of Ace-/- mice, the ectodomain of sACE, which corresponded to only the secreted form of ACE. Our results demonstrated that the secreted form of ACE could normalize kidney functions and RAS integrity, growth and development of Ace-/- mice, but not their blood pressure. This study clearly demonstrates that the secreted form of ACE cannot replace the tissue-bound ACE for maintaining normal blood pressure; a suitable balance between the tissue-bound and the soluble forms of ACE is essential for maintaining all physiological functions of ACE.

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A Small Region in the Angiotensin-Converting Enzyme Distal Ectodomain Is Required for Cleavage-Secretion of the Protein at the Plasma Membrane

Cited by 4 publications

References 52 publications

Kinetic and structural characterization of amyloid‐β peptide hydrolysis by human angiotensin‐1‐converting enzyme

Kinetic and structural characterization of amyloid‐β peptide hydrolysis by human angiotensin‐1‐converting enzyme

Mast cell chymase limits the cardiac efficacy of Ang I–converting enzyme inhibitor therapy in rodents

Tissue-Specific Expression of Transgenic Secreted ACE in Vasculature Can Restore Normal Kidney Functions, but Not Blood Pressure, of Ace-/- Mice

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