B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction.

Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.

Effects of rubber elongation factor and small rubber particle protein from rubber-producing plants on lipid metabolism in Saccharomyces cerevisiae.

The common proteins rubber elongation factor (REF) and small rubber particle protein (SRPP) are associated with Hevea brasiliensis (Hb) rubber particles. They are involved in the stability of rubber particles and natural rubber biosynthesis. Recently, we cloned cDNAs encoding REF and SRPP from laticifers of Ficus carica (Fc). In the present study, we overexpressed REF/SRPPs (HbREF, FcREF, and FcSRPP) in Saccharomyces cerevisiae in anticipation of future rubber biosynthesis in recombinant yeast. The proteins were localized in the endoplasmic reticulum and lipid droplets (LDs), and affected LD morphology. Furthermore, their overexpression resulted in an accumulation of neutral lipids and a decrease in yeast cell size. This suggests that REF/SRPPs affect lipid metabolism and lead to a decline in the phospholipid content of yeast. We also found that expression of these proteins induced accumulation of steryl esters and triacylglycerols in yeast. This suggests that the coexpression of REF/SRPPs with key enzymes for the biosynthesis of target lipids in yeast is a promising way of increasing production of important lipids like triacylglycerols and terpenes, and that a protein complex consisting of cis-prenyltransferase (CPT), CPT-like protein, and REF/SRPPs for rubber biosynthesis could be reconstituted on yeast lipid droplets.

Cloning and Aggregation Characterization of Rubber Elongation Factor and Small Rubber Particle Protein from Ficus carica.

Rubber elongation factor (REF) and small rubber particle protein (SRPP) are major latex proteins harvested from Hevea brasiliensis (the rubber tree; HbREF and HbSRPP, respectively). Their amino acid sequences exhibit high homology with each other. In the present study, we cloned two cDNAs encoding REF/SRPP-family proteins (FcREF/SRPP-1 and -2) from the laticifers of Ficus carica (fig tree). The amino acid sequences of these proteins showed high homology not only with each other but also with HbREF and HbSRPP. Recombinant FcREF/SRPP-1 and -2 were expressed in E. coli, and their aggregation properties were examined using a Congo red binding assay, agarose gel electrophoresis, and transmission electron microscopy. FcREF/SRPP-1 formed fibrils when incubated in PBS, and grew to micrometer-sized amorphous aggregates that precipitated rapidly. These aggregation properties of FcREF/SRPP-1 are quite similar to those of HbREF, although the growth rate and size of FcREF/SRPP-1 aggregates were inferior to those of HbREF. FcREF/SRPP-2 also formed aggregates during the incubation, but they did not precipitate, as has been reported for HbSRPP. Our results suggest that FcREF/SRPP-1 and -2 correspond to HbREF and HbSRPP, respectively. These aggregation properties could provide useful benchmarks for classifying REF/SRPP-family proteins as REF or SRPP.