Intracellular Peptides in Cell Biology and Pharmacology

Abstract

Intracellular peptides are produced by proteasomes following degradation of nuclear, cytosolic, and mitochondrial proteins, and can be further processed by additional peptidases generating a larger pool of peptides within cells. Thousands of intracellular peptides have been sequenced in plants, yeast, zebrafish, rodents, and in human cells and tissues. Relative levels of intracellular peptides undergo changes in human diseases and also when cells are stimulated, corroborating their biological function. However, only a few intracellular peptides have been pharmacologically characterized and their biological significance and mechanism of action remains elusive. Here, some historical and general aspects on intracellular peptides' biology and pharmacology are presented. Hemopressin and Pep19 are examples of intracellular peptides pharmacologically characterized as inverse agonists to cannabinoid type 1 G-protein coupled receptors (CB1R), and hemopressin fragment NFKF is shown herein to attenuate the symptoms of pilocarpine-induced epileptic seizures. Intracellular peptides EL28 (derived from proteasome 26S protease regulatory subunit 4; Rpt2), PepH (derived from Histone H2B type 1-H), and Pep5 (derived from G1/S-specific cyclin D2) are examples of peptides that function intracellularly. Intracellular peptides are suggested as biological functional molecules, and are also promising prototypes for new drug development.

Keywords: cancer; drug discovery; endocannabinoid; epilepsy; insulin resistance; intracellular peptides; obesity; proteasome.

Figure 1

Pharmacological and molecular characterization of cannabinoids in cannabinoid type 1 G-protein coupled receptors (CB1R)-associated epilepsy treatment. (A–D) adult male C57BL/6J wild-type mice weighting approximately 24 g were kept in animal room with a controlled temperature (22 ± 1 °C) and a light–dark cycle of 12 h, and water and feed was supplied ad libitum. The number of animals used was the minimum necessary to obtain statistically significant results and they were maintained and used in accordance to the guidelines of the National Council for Control of Animal Experiments (CONCEA), following international norms of animal care and maintenance (Ethics protocol number ICB/USP 5100050218). Induction of epilepsy was by intraperitoneal administration of pilocarpine hydrochloride (Merck, SP, Brazil; 320 mg/kg) dissolved in 0.9% sterile saline. Hemopressin, NFKF, rimonabant, and cannabidiol (kindly provided by Professor Raphael Mechoulam, Hebrew University of Jerusalem, Israel, to Professor Francisco Guimarães, Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, SP, Brazil) were administered 10 min prior to the administration of pilocarpine. Time to salivation, time to the first motor seizure, number of seizure events, and death were quantified from 0 to 30 min after pilocarpine injection, as previously described [102]. (E) Hemopressin or NFKF (1 mg/mL, 500 µL) were freshly prepared in sterile water, or incubated at −20 °C for 24 h or at 100 °C for 15 min, and the peptides that remained in solution were compared by high performance liquid chromatography (HPLC), as previously described [103]. All biological results are expressed as the means ± standard error of the mean (SEM). The statistical comparisons were performed using Student’s t-test or analysis of variance (ANOVA), followed by ad-hoc Tukey’s test. Probability less than 0.05 was considered as statistically significant (p < 0.05). * p < 0.05 vs. control, # p < 0.05 vs. HP, ** p < 0.05 vs. control, *** p < 0.001 vs. control. Data were statistically analyzed with GraphPad Prism software (GraphPad Software Inc, San Diego, CA, USA). (F–I) Molecular docking studies using the crystallographic structure of CB1R available at the Protein Data Bank (5TGZ). NFKF (panel I) has higher binding affinity (higher Goldscore) for CB1R than AM6538 (panel F), cannabidiol (G) and rimonabant panel (H).

Figure 2

Hypothetical model of Pep5 generation by the ubiquitin–proteasome system (UPS). During G1 phase, cyclin D2 forms a complex with cyclin-dependent kinases (CDKs) to regulate several processes. At the G1/S boundary, the cyclin D2 levels decrease after ubiquitination (Ub) and proteasome degradation. As a result of this degradation, several peptides are formed and released in the intracellular environment. Some of the peptides will be entirely degraded by peptidases whereas some peptides (e.g., Pep5) will remain in the cells to participate in biological processes.

Figure 3

Schematic representation of isotopic-labeling of peptide-containing cellular extracts isolated in different phases of the cell cycle of the Hela cell line. Peptides were extracted from the asynchronous cells and labeled with D0-TMAB, whereas those synchronized in S, G2/M, and G0/G1 were, respectively, labeled with D3-TMAB, D9-TMAB, or D12-TMAB.

Figure 4

Pep5-cpp leading to cell death seems a combination of events. Once Pep5 is bound to a cell-penetrating peptide, it will carry the cyclin D2 fragment to intracellular compartments such as the cytoplasm and nucleus. (1) Pep5-cpp binds to chloride intracellular channel protein 1 (CLIC1) and (2) Plectin, leading to (3) actin cytoskeleton disorganization and (4) ERK1/2 phosphorylation. The sustained increase in ERK1/2 phosphorylation may contribute to the activation of caspases 3/7 and 9, culminating in the induction of apoptosis. On the other hand, Pep5-cpp also seems to bind to (5) proteasome components causing its inhibition, which in turn can cause protein accumulation in both the cytosol and nucleus, generating signals that will induce programmed necrosis (necroptosis).