Leandro M. Castro

Universidade Estadual Paulista (Unesp)

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Emer S. Ferro

Universidade de São Paulo

Lloyd D. Fricker

Albert Einstein College of Medicine

Vanessa Rioli

Instituto Butantan

Fábio C. Gozzo

Hospital de Clínicas da Unicamp

Lilian C. Russo

Universidade de São Paulo

Dânia Emi Hamassaki‐Britto

Universidade de São Paulo

Vitor Oliveira

New York Medical College

Sayani Dasgupta

Albert Einstein College of Medicine

Cláudia Neves Correa

Universidade Estadual Paulista (Unesp)

Alexandre Hiroaki Kihara

Universidade Federal do ABC

Cooperative Institutions

Universidade de São Paulo

122

Universidade Federal de São Paulo

Instituto Butantan

Universidade Estadual Paulista (Unesp)

Universidade Estadual de Campinas (UNICAMP)

Institute of Biomedical Science

Albert Einstein College of Medicine

Universidade Brasil

Harvard University

Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo

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Intracellular peptides: From discovery to function

EuPA Open Proteomics (2014)

Emer S. Ferro Vanessa Rioli Leandro M. Castro Lloyd D. Fricker

Peptidomics techniques have identified hundreds of peptides that are derived from proteins present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular peptides to distinguish them from secretory pathway peptides that function primarily outside of the cell. The proteasome and thimet oligopeptidase participate in the production and metabolism of intracellular peptides. Many of the intracellular peptides are common among mouse tissues and human cell lines analyzed and likely to perform a variety of functions within cells. Demonstrated functions include the modulation of signal transduction, mitochondrial stress, and development; additional functions will likely be found for intracellular peptides.

10.1016/j.euprot.2014.02.009

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Citations (59)

Neurolysin Knockout Mice Generation and Initial Phenotype Characterization

Journal of Biological Chemistry (2014)

Diogo Manuel Lopes de Paiva Cavalcanti Leandro M. Castro José Cesar Rosa Neto Marília Seelaender Rodrigo Xavier das Neves

The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln KO mice have increased glucose tolerance, insulin sensitivity, and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semiquantitative peptidomic analysis suggests an increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity. The oligopeptidase neurolysin (EC 3.4.24.16; Nln) was first identified in rat brain synaptic membranes and shown to ubiquitously participate in the catabolism of bioactive peptides such as neurotensin and bradykinin. Recently, it was suggested that Nln reduction could improve insulin sensitivity. Here, we have shown that Nln KO mice have increased glucose tolerance, insulin sensitivity, and gluconeogenesis. KO mice have increased liver mRNA for several genes related to gluconeogenesis. Isotopic label semiquantitative peptidomic analysis suggests an increase in specific intracellular peptides in gastrocnemius and epididymal adipose tissue, which likely is involved with the increased glucose tolerance and insulin sensitivity in the KO mice. These results suggest the exciting new possibility that Nln is a key enzyme for energy metabolism and could be a novel therapeutic target to improve glucose uptake and insulin sensitivity.

Gluconeogenesis

Oligopeptidase

Catabolism

GLUT4

10.1074/jbc.m113.539148

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Citations (48)

Chiasmocleis napolii sp. nov.: a new species of Chiasmocleis Méhelÿ, 1904 (Anura: Microhylidae) from the Atlantic Forest, Northeastern Brazil.

Zenodo (CERN European Organization for Nuclear Research) (2018)

Carlos Alberto Gonçalves Cruz Pedro Henrique A. G. Moura Marcelo Gomes de Lima Leandro M. Castro Ivan Nunes

Nexus file of the 16S rRNA mtDNA matrix used for phylogenetic analysis and MEGA file of matrix used for calculating genetic distances.

Atlantic forest

10.5281/zenodo.2525730

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Peptidomic Analysis of Human Cell Lines

Journal of Proteome Research (2011)

Julia S. Gelman Juan Sironi Leandro M. Castro Emer S. Ferro Lloyd D. Fricker

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEK293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the P1 position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

HEK 293 cells

Cleavage (geology)

10.1021/pr100952f

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Citations (69)

Translocation of cytochrome C to the cytoplasm and nucleus in non-apoptotic cells

Free Radical Biology and Medicine (2007)

Luiz C. Godoy Catalina Arango Pinedo Leandro M. Castro Simone Cardaci Christopher M. Schonhoff

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14‐3‐3 epsilon modulates the stimulated secretion of endopeptidase 24.15

Journal of Neurochemistry (2005)

Flavia Carreño Camila N. Goñi Leandro M. Castro Emer S. Ferro

Abstract Endopeptidase 24.15 (ep24.15: EC3.4.24.15), a secreted protein involved in peptide metabolism, is unusual in that it does not contain a signal peptide sequence. In this work, we describe the physical interaction between ep24.15 and 14‐3‐3 epsilon, one isoform of a family of ubiquitous phosphoserine/threonine‐scaffold proteins that organizes cell signaling and is involved in exocytosis. The interaction between ep24.15 and 14‐3‐3 epsilon increased following phosphorylation of ep24.15 at Ser 644 by protein kinase A (PKA). The co‐localization of ep24.15 and 14‐3‐3 epsilon was increased by exposure of HEK293 cells (human embryonic kidney cells) to forskolin (10 µ m ). Overexpression of 14‐3‐3 epsilon in HEK293 cells almost doubled the secretion of ep24.15 stimulated by A23187 (7.5 µ m ) from 10%[1.4 ± 0.24 AFU/(min 10 6 cells)] to 19%[2.54 ± 0.24 AFU/(min 10 6 cells)] ( p < 0.001) of the total intracellular enzyme activity. Treatment with forskolin had a synergistic effect on the A23187‐stimulated secretion of ep24.15 that was totally blocked by the PKA inhibitor KT5720. The ep24.15 point mutation S644A reduced the co‐localization of ep24.15 and 14‐3‐3 in stably transfected HEK293 cells. Indeed, secretion of the ep24.15 S644A mutant from these cells was only slightly stimulated by A23187 and insensitive to forskolin, in contrast to that of the wild type enzyme. Together, these data suggest that prior interaction with 14‐3‐3 is an important step in the unconventional stimulated secretion of ep24.15.

HEK 293 cells

Endopeptidase

Phosphoserine

10.1111/j.1471-4159.2004.02967.x

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Citations (34)

Análise imuno-histoquímica da população de linfócitos TCD4+, linfócitos TCD8+, macrófagos CD68+ e células NK em pulmões de necrópsias de pacientes HIV+/AIDS com quadro de pneumonia por pneumocystis carinii

Resumos (2002)

Leandro M. Castro Carla Pagliari E. R. Fernandes Maria Irma Seixas Duarte

CD68

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Revealing Natural Intracellular Peptides in Gills of Seahorse Hippocampus reidi

Biomolecules (2023)

Cláudia Neves Correa Louise Oliveira Fiametti Gabriel Marques de Barros Leandro M. Castro

The seahorse is a marine teleost fish member of the Syngnathidae family that displays a complex variety of morphological and reproductive behavior innovations and has been recognized for its medicinal importance. In the Brazilian ichthyofauna, the seahorse Hippocampus reidi is among the three fish species most used by the population in traditional medicine. In this study, a protocol was performed based on fast heat inactivation of proteases plus liquid chromatography coupled to mass spectrometry to identify native peptides in gills of seahorse H. reidi. The MS/MS spectra obtained from gills allowed the identification of 1080 peptides, of which 1013 peptides were present in all samples and 67 peptide sequences were identified in an additional LC-MS/MS run from an alkylated and reduced pool of samples. The majority of peptides were fragments of the internal region of the amino acid sequence of the precursor proteins (67%), and N- and C-terminal represented 18% and 15%, respectively. Many peptide sequences presented ribosomal proteins, histones and hemoglobin as precursor proteins. In addition, peptide fragments from moronecidin-like protein, described with antimicrobial activity, were found in all gill samples of H. reidi. The identified sequences may reveal new bioactive peptides.

Seahorse

10.3390/biom13030433

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Citations (1)

Author Index

Journal of Biological Chemistry (2014)

María Abad Scott Abrams Akiyama Chie Akiyama Tomohito Amigo -Jiménez

10.1016/s0021-9258(20)38577-x

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A Novel Intracellular Peptide Derived from G1/S Cyclin D2 Induces Cell Death

Journal of Biological Chemistry (2014)

Christiane B. de Araujo Lilian C. Russo Leandro M. Castro Fábio Luís Forti Elisabete Rodrigues do Monte

Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides. Intracellular peptides are constantly produced by the ubiquitin-proteasome system, and many are probably functional. Here, the peptide WELVVLGKL (pep5) from G1/S-specific cyclin D2 showed a 2-fold increase during the S phase of HeLa cell cycle. pep5 (25–100 μm) induced cell death in several tumor cells only when it was fused to a cell-penetrating peptide (pep5-cpp), suggesting its intracellular function. In vivo, pep5-cpp reduced the volume of the rat C6 glioblastoma by almost 50%. The tryptophan at the N terminus of pep5 is essential for its cell death activity, and N terminus acetylation reduced the potency of pep5-cpp. WELVVL is the minimal active sequence of pep5, whereas Leu-Ala substitutions totally abolished pep5 cell death activity. Findings from the initial characterization of the cell death/signaling mechanism of pep5 include caspase 3/7 and 9 activation, inhibition of Akt2 phosphorylation, activation of p38α and -γ, and inhibition of proteasome activity. Further pharmacological analyses suggest that pep5 can trigger cell death by distinctive pathways, which can be blocked by IM-54 or a combination of necrostatin-1 and q-VD-OPh. These data further support the biological and pharmacological potential of intracellular peptides.

HeLa

Cyclin A

10.1074/jbc.m113.537118

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Citations (46)