Identification of the recognition sequence and target proteins for DJ-1 protease

DJ-1, the product of familial Parkinson's disease gene and an oncogene, is a cysteine protease which plays a role in anti-oxidative stress reaction. In this study, we identified the recognition sequence for DJ-1 protease by using recombinant DJ-1 and a peptide library. Protease activity of DJ-1 lacking C-terminal alpha-helix (DJ-1 Delta H9) was stronger than that of full-sized DJ-1, and the most susceptible sequence digested by DJ-1 Delta H9 was valine-lysine-valine-alanine (VKVA) under the optimal conditions of pH 5.5 and 0 mM NaCl. Divalent ions, especially Cu2+, were inhibitory to DJ-1's protease activity. c-abl oncogene 1 product (ABL1) and kinesin family member 1B (KIF1B) containing VKVA were digested by DJ-1 Delta H9. Structured summary of protein interactions: DJ-1 cleaves IUF1B by enzymatic study (View interaction) DJ-1 cleaves ABLI by enzymatic study (View interaction) (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

20 kDa PEGylated Adrenomedullin as a New Therapeutic Candidate for Inflammatory Bowel Disease

Human adrenomedullin (AM), a hypotensive peptide, also has anti-colitis activity. We prepared a polyethylene glycol (PEG) ylated form of AM through the conjugation of PEG-AM (1–15) and AM (15–52). Highly pure monomeric 20 kDa PEG-AM (20kPEG-AM) stimulated cyclic adenosine monophosphate production in HEK-293 cells stably expressing the type 1 AM receptor in a dose-dependent manner. The half-life of 20kPEG-AM was 7.4 h following subcutaneous administration in mice. We assessed the anti-colitis effect of subcutaneous 20kPEG-AM administration in the dextran sodium sulfate murine colitis model. Single and double subcutaneous injection of 20kPEG-AM significantly reduced total inflammation scores. These results suggest that 20kPEG-AM is a promising therapeutic candidate for the treatment of human inflammatory bowel diseases

Absolute Quantitation of Low Abundance Plasma APL1β peptides at Sub-fmol/mL Level by SRM/MRM without Immunoaffinity Enrichment

Selected/multiple reaction monitoring (SRM/MRM) has been widely used for the quantification of specific proteins/peptides, although it is still challenging to quantitate low abundant proteins/peptides in complex samples such as plasma/serum. To overcome this problem, enrichment of target proteins/peptides is needed, such as immunoprecipitation; however, this is labor-intense and generation of antibodies is highly expensive. In this study, we attempted to quantify plasma low abundant APLP1-derived Aβ-like peptides (APL1β), a surrogate marker for Alzheimer’s disease, by SRM/MRM using stable isotope-labeled reference peptides without immunoaffinity enrichment. A combination of Cibacron Blue dye mediated albumin removal and acetonitrile extraction followed by C₁₈-strong cation exchange multi-StageTip purification was used to deplete plasma proteins and unnecessary peptides. Optimal and validated precursor ions to fragment ion transitions of APL1β were developed on a triple quadruple mass spectrometer, and the nanoliquid chromatography gradient for peptide separation was optimized to minimize the biological interference of plasma. Using the stable isotope-labeled (SI) peptide as an internal control, absolute concentrations of plasma APL1β peptide could be quantified as several hundred amol/mL. To our knowledge, this is the lowest detection level of endogenous plasma peptide quantified by SRM/MRM

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

Brain p3-Alcβ peptide restores neuronal viability impaired by Alzheimer's amyloid β-peptide

We propose a new therapeutic strategy for Alzheimer's disease (AD). Brain peptide p3-Alc beta 37 is generated from the neuronal protein alcadein beta through cleavage of gamma-secretase, similar to the generation of amyloid beta (A beta) derived from A beta-protein precursor/APP. Neurotoxicity by A beta oligomers (A beta o) is the prime cause prior to the loss of brain function in AD. We found that p3-Alc beta 37 and its shorter peptide p3-Alc beta 9-19 enhanced the mitochondrial activity of neurons and protected neurons against A beta o-induced toxicity. This is due to the suppression of the A beta o-mediated excessive Ca2+ influx into neurons by p3-Alc beta. Successful transfer of p3-Alc beta 9-19 into the brain following peripheral administration improved the mitochondrial viability in the brain of AD mice model, in which the mitochondrial activity is attenuated by increasing the neurotoxic human A beta 42 burden, as revealed through brain PET imaging to monitor mitochondrial function. Because mitochondrial dysfunction is common in the brain of AD patients alongside increased A beta and reduced p3-Alc beta 37 levels, the administration of p3-Alc beta 9-19 may be a promising treatment for restoring, protecting, and promoting brain functions in patients with AD

20 kDa PEGylated Adrenomedullin as a New Therapeutic Candidate for Inflammatory Bowel Disease

Human adrenomedullin (AM), a hypotensive peptide, also has anti-colitis activity. We prepared a polyethylene glycol (PEG) ylated form of AM through the conjugation of PEG-AM (1–15) and AM (15–52). Highly pure monomeric 20 kDa PEG-AM (20kPEG-AM) stimulated cyclic adenosine monophosphate production in HEK-293 cells stably expressing the type 1 AM receptor in a dose-dependent manner. The half-life of 20kPEG-AM was 7.4 h following subcutaneous administration in mice. We assessed the anti-colitis effect of subcutaneous 20kPEG-AM administration in the dextran sodium sulfate murine colitis model. Single and double subcutaneous injection of 20kPEG-AM significantly reduced total inflammation scores. These results suggest that 20kPEG-AM is a promising therapeutic candidate for the treatment of human inflammatory bowel diseases.Citation: Miki, G.; Kuroishi, N.; Tokashiki, M.; Nagata, S.; Tamura, M.; Yoshiya, T.; Yoshizawa-Kumagaye, K.; Ashizuka, S.; Kato, J.; Yamasaki, M.; et al. 20 kDa PEGylated Adrenomedullin as a New Therapeutic Candidate for Inflammatory Bowel Disease. Gastrointest. Disord. 2020, 2, 366-377. https://doi.org/10.3390/gidisord204003