Page 12 - PR 2014 2016 04 Biotechnology

98 Biotechnology | Progress Report

Structural analyses of site of C-domain of angiotensin-converting
soluble human protein enzyme possesses enzymatic activity and is

inhibited by Lisinopril. This peptide can be
The L10 ribosomal protein (RPL10) plays a used to test new inhibitors and C-domain of
role in the binding of the 60 S and 40 S ri - angiotensin-converting enzyme substrates
bosomal subunits and in mRNA translation. because this peptide is easy to produce and
The evidence indicates that RPL10 also has this has proven efficient link with these mol-
multiple extra-ribosomal functions, including ecules (“Structural characterization and enzy-
tumor suppression and its overexpression matic activity of the recombinant Ala959 to
osteoblasts exhibit a cell autonomous alter- Ser1066 region of human ACE”). Osteoblasts
ation that lead to increased mineralization in are specialized fibroblasts that secrete and
vitro. We successfully cloned and expressed mineralize the bone matrix, and there is little
full-length human RPL10 (hRPL10) protein information on the fate and potential thera-
and isolated inclusion bodies that had been peutic efficacy of low-dose gamma-irradiation
formed under mild growth conditions. Af- in the formation of mineralization nodules
ter the hRPL10 purification using a two-step in the osteoblast culture. Our first results of
process of non-denaturing protein extraction low dose irradiated osteoblasts showed an in-
from pelleted inclusion bodies, we studied crease in the number of mineralized nodules.
the characteristics of this protein using cir-
cular dichroism spectroscopy and by moni-
toring the changes induced by the presence
or absence of zinc ions using fluorescence Biological effects of
spectrometry. The results suggested that the ionizing radiation
strategy used to obtain hRPL10 is simple and
could be applied to obtaining other proteins Biological effects of ionizing radiation in
that are susceptible to degradation (“A simple aqueous solution, produces several highly
strategy for the purification of native recom- reactive species. The most important are hy-
binant full-length human RPL10 protein from droxyl radical and hydrated electron. These
inclusion bodies”). Angiotensin-converting products interact with peptides and proteins
enzyme catalyzes the conversion of angio- causing several modifications such as frag-
tensin I to the vasoconstrictor angiotensin mentation, aggregation or oxidation, which
II and the hydrolysis of bradykinin (BK). Hu- are responsible for detoxification or even few
man somatic angiotensin-converting enzyme modifications on proteins. These properties of
has two homologous domains (N and C) that ionizing radiation make it a good tool to im-
share 60% identity, and the catalytic site of prove antiserum production and vaccination
the C-domain exhibits three-fold greater ac- process. Additionally, some substances called
tivity than the N-domain in the hydrolysis scavenger can be used to modulate these ef-
of angiotensin I in vivo. The catalytic site of fects. It was found that the irradiated protein
C-domain of angiotensin-converting enzyme could be selectively incorporated to the cells,
peptide was expressed in a bacterial system, due to specific receptor for oxidized protein,
and its purification was performed in one the scavenger receptors. This increased uptake
step. Structural analysis by circular dichroism could also result in better antigen presentation
and fluorescence confirmed that the puri- and high immune response, either humoral, as
fied protein is correctly folded, and catalytic demonstrated with purified crotoxin or cellular,

Instituto de Pesquisas Energéticas e Nucleares