Intracellular Peptides: from Discovery to Function

e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151

Available online at www.sciencedirect.com

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journal homepage: http://www.elsevier.com/locate/euprot

Intracellular peptides: From discovery to function

a,∗ b a,c d,∗∗

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

Department of Pharmacology, Biomedical Sciences Institute, University of São Paulo, São Paulo, SP, Brazil

Special Laboratory of Applied Toxicology-CeTICS, Butantan Institute, São Paulo, SP, Brazil

Department of Biophysics, Federal University of São Paulo, São Paulo, SP, Brazil

Department of Molecular Pharmacology and Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA

a r t i c l e i n f o a b s t r a c t

Article history: Peptidomics techniques have identiﬁed hundreds of peptides that are derived from proteins

Received 8 October 2013 present mainly in the cytosol, mitochondria, and/or nucleus; these are termed intracellular

Received in revised form peptides to distinguish them from secretory pathway peptides that function primarily out-

29 November 2013 side of the cell. The proteasome and thimet oligopeptidase participate in the production and

Accepted 14 February 2014 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

Keywords: within cells. Demonstrated functions include the modulation of signal transduction, mito-

Peptides chondrial stress, and development; additional functions will likely be found for intracellular

Cell signaling peptides.

Protein–protein interaction Association (EuPA). This is an open access article under the CC BY-NC-ND license Receptors (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Mass spectrometry

with a half life of several seconds [2,3]. Some of the pep-

1. Historical perspectives

tides produced by the proteasome are protected from cytosolic

aminopeptidases by transport into the endoplasmic reticulum

Ever since the discovery of the proteasome, a multisubunit

(ER) by a peptide transporter. Once in the ER, if the peptide is

complex that converts proteins into peptides, it was recog-

the correct size and contains the appropriate amino acids in

nized that peptides would transiently exist inside of cells

key positions, the peptide binds to major histocompatibility

(Fig. 1). The formation and degradation of intracellular pep-

complex I (MHC-I) and is transported to the cell surface where

tides is as complex as the production and degradation of

it serves in antigen presentation [2]. If the peptide is too long

microRNA (Fig. 1). Intracellular peptides typically range in size

to bind to MHC-I, an ER resident aminopeptidase trims the

from 2 to 21 amino acids, with the average size of the protea-

peptide until it is either the correct length and able to bind to

some degradation products 10–12 amino acids in length [1].

MHC-I, or if unable to bind to MHC1, the aminopeptidase con-

The conventional view is that the proteasome-produced pep-

tinues until the peptide is degraded. Ultimately, only a very

tides are rapidly broken down by cytosolic aminopeptidases

small fraction of intracellular peptides (e.g. one peptide of 9–11

∗

Corresponding author at: Department of Pharmacology, Support Center for Research in Proteolysis and Cell Signaling (NAPPS), Room 315,

Biomedical Sciences Institute, University of São Paulo, Av. Prof. Lineu Prestes 1524, São Paulo, SP 05508-000, Brazil. Tel.: +55 11 30917493.

∗∗

Corresponding author at: Department of Molecular Pharmacology, Albert Einstein College of Medicine, 1300 Morris Park Avenue F248,

Bronx, NY 10461, USA.

E-mail addresses: [email protected] (E.S. Ferro), [email protected] (L.D. Fricker).

http://dx.doi.org/10.1016/j.euprot.2014.02.009

article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

144 e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151

Fig. 1 – Comparison of the key steps in the formation and degradation of microRNA and intracellular peptides. Micro RNA

(miRNA) is produced from primary transcripts (pri-mRNA) by a series of nucleases: Drosha, Pasha, and Dicer. Once the

miRNA duplexes dissociate to form the miRNA, it is able to form an RNA-induced silencing complex that contains many

associated proteins including members of the argonaute protein family. Degradation of miRNA–RNA complex is mediated by

ribonucleases. Intracellular peptides are primarily produced by the proteasome although other proteases such as calpains,

caspases, and mitochondrial enzymes are also known to generate intracellular peptides. There are several intracellular

peptidases that cleave moderately sized peptides, which are not able to directly cleave proteins. These oligopeptidases

include endopeptidase 24.15 (also known as thimet oligopeptidase), neurolysin (also known as endopeptidase 24.16),

insulin-degrading enzyme, prolyl oligopeptidase, and nardilysin. These enzymes have different substrate speciﬁcities such

that they each produce distinct patterns of intracellular peptides from the proteasome-produced peptides. There is evidence

that some of the intracellular peptides are biologically active. Final degradation of the intracellular peptides is mediated by

aminopeptidases, including tripeptidyl peptidase II, leucine aminopeptides, puromycin-sensitive aminopeptidase, and

bleomycin hydrolase. In addition to the formation of intracellular peptides from proteins, it is also possible that intracellular

peptides are produced directly from RNA with short open reading frames [74,75] or from defective ribosome products [76].

amino acids for each protein) end up on cell surface, and until each other using peptides produced from cytosolic pro-

recent peptidomics analyses detected a large number of intra- teins. Some peptides were isolated from mammalian brains

cellular peptides (described below), only a small number of based on bioactive properties, but when these molecules

intracellular peptides were known. were found to represent fragments of intracellular proteins,

One of the best-studied intracellular peptides is the yeast there was little enthusiasm amongst the scientiﬁc commu-

Saccharomyces cerevisiae a-factor, which functions as a mating nity. Examples of bioactive peptides from cytosolic proteins

pheromone [4]. Yeast has two peptide mating factors, named include diazepam binding inhibitor (DBI) from acyl-coA bind-

alpha-factor and a-factor. Alpha-factor is produced within the ing protein and hippocampal cholinergic neurostimulating

secretory pathway, much like mammalian peptide hormones peptide (HCNP) from phosphatidylethanolamine-binding pro-

(although with different processing enzymes), stored in vesi- tein [5–7]. During the 1980s and 1990s while the above studies

cles, and secreted when the vesicles fuse with the cellular were on-going, a number of cellular endopeptidases were dis-

membrane. In contrast, a-factor is produced within the cytosol covered and characterized (Fig. 1). Some of these enzymes,

by a series of steps involving lipid attachment (prenylation), such as endopeptidase 24.15 (also named endo-oligopeptidase

N-terminal proteolytic cleavages by Ste24p and Axl1p, and A and thimet oligopeptidase), are able to convert neuro-

transport from the cytosol to the extracellular space by Ste6p peptides into smaller fragments; in some cases this conversion

[4]. The secreted a-factor binds to a speciﬁc receptor (Ste3p) causes the neuropeptide to be inactive, while in other cases

and stimulates mating. the product is also biologically active but with differing recep-

Despite the precedence for cytosolic peptides function- tor speciﬁcity [8–11]. In the latter cases, the endopeptidase

ing in plasma membrane receptor-mediated cell-cell signaling was thought to play a modulatory role, rather than simply

that was provided from studies on yeast a-factor, there has activating or inactivating the peptide. While the extracellu-

been resistance to the idea that mammalian cells signal lar processing of neuropeptides may represent a bona ﬁde

e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151 145

function for endopeptidase 24.15 and related enzymes, the ATP-dependent Clp proteases. The combined expression of

ﬁnding that the vast majority of the enzyme is present in these proteins represents the cell response toward the damage

the cytosol and/or nucleus suggested other functions for these produced by irreversible aggregates and protein misfolding

enzymes [12]. One suggested function was in the processing affecting cell functions and survival. In general, this response

of peptides post-proteasome for eventual binding to MHC-I, as is elicited to maintain protein homeostasis by increasing the

part of the antigen presentation machinery [13]. However, the expression of heat shock proteins (chaperones) to assist the

substrate speciﬁcity did not entirely ﬁt with this scheme, as process of protein folding. Signal peptides generated by the

the majority of MHC-I antigens evaluated in vitro were not good mitochondrial ATP-dependent proteolytic complex can acti-

substrates of endopeptidase 24.15 [14]. Thus, the function of vate the cell genome, providing a mechanism of intercellular

endopeptidase 24.15 and related intracellular oligopeptidases communication among mitochondria, cytosol and nucleus

was not clear, and a logical function would be a role in the post- [24].

proteasome processing of intracellular peptides that served as

signaling molecules within cells (Fig. 1) [15]. The intracellular

peptides bear a resemblance to microRNAs that are produced

by selective intracellular enzymes and ultimately regulate pro- 3. Intracellular proteolytic generation of

tein function (Fig. 1). peptides

In eukaryotic cells, most proteins destined for degradation

2. Peptide and protein interactions in cell are initially tagged with a polyubiquitin chain in an energy-

communication dependent process, and then digested to peptides by the 26S

proteasome, a large proteolytic complex involved in the regu-

Many studies have used synthetic peptides to perturb cel- lation of cell division, gene expression and other key processes

lular functions. For example, peptides capable of inhibiting [25–27]. In one study it was estimated that in eukaryotes, the

the interactions between kinases and their anchoring proteins proteasome converts 30–90% of newly synthesized proteins

[16] were rationally designed, synthesized and shown to regu- into peptides within minutes [28]. Therefore, the concomitant

late kinase-mediated functions [17,18]. Kaneto and co-workers action of proteasomes and other proteolytic systems [29,30] in

demonstrated that c-Jun N-terminal kinase is inhibited by the cytosol, nucleus and mitochondria suggests a continuous

a peptide derived from a kinase-interacting protein, preven- formation and release of peptides within cells [2,31,32].

ting the access of the kinase to its substrate c-Jun [19]. When Following the proteasome, peptides can be further cleaved

a cell-permeable version of this peptide was injected into by cytosolic oligopeptidases (Fig. 1). Over the past decades,

mice, a signiﬁcant improvement in insulin resistance was many peptidases have been isolated and characterized [33–39].

observed, with consequent improvement of glucose tolerance, Although most of these enzymes are present in the extracel-

in a model of type 2 diabetes. Peptides derived from A-kinase lular medium—an essential prerequisite for their association

anchoring protein have been used to alter subcellular local- to the physiological metabolism of neuropeptides—many of

ization of protein kinase A [20]. A peptide named inhibitory them predominate in the intracellular milieu. Some of the

peptide, was characterized as a highly potent and speciﬁc intracellular enzymes can process both proteins and peptides.

substrate competitive inhibitor of Calmodulin-Dependent Pro- For example, tripeptidyl-peptidase II primarily cleaves three

tein Kinase II [21], which plays important roles in controlling amino acid residues from the N-terminal of peptides/proteins,

a variety of cellular functions in the Central Nervous Sys- and also functions as an endopeptidase [35,39,40]. Sev-

tem [22,23]. All of these studies used synthetic peptides, and eral other cytosolic endopeptidases are known for their

although the sequences were based on protein sequences, it selectivity for oligopeptides and inability to directly cleave

was not thought that these peptides (or similar ones) existed proteins into peptides. Examples include insulin-degrading

in vivo. enzyme (IDE), endopeptidase 24.15 (thimet oligopeptidase),

Endogenous bioactive peptides have been shown to play endopeptidase 24.16 (neurolysin), and prolyl oligopeptidase

a role in the maintenance of mitochondrial protein stability [33,34,36–38,41,42]. Although oligopeptidases are not likely to

[24]. In mitochondria, proteases degrade proteins into pep- initiate cleavages of proteins due to their restrictions on sub-

tides that are exported from the mitochondrial matrix into the strate size, such enzymes contribute to the production of

intermembrane space by ABC transporters and reach the cyto- peptides from intermediates generated by the proteasome

sol by passive diffusion. Recently, Haynes et al. demonstrated system or other intracellular proteases.

by genetic analysis of Caenorhabilis elegans that under stress The various cytosolic oligopeptidases do not appear to be

of mitochondrial protein misfolding, signals are emitted from degradative enzymes, but instead have fairly limited sub-

the mitochondrial matrix to the nucleus by the bZIP protein, strate speciﬁcities. For example, endopeptidase 24.15 is able

which regulates nuclear-encoded mitochondrial chaperone to cleave only a subset of the peptides present in the cytosol;

genes [24]. Consequently, the organelle can react against the some are much better substrates than others [38]. Further-

loss of thermodynamic stability and the propensity of proteins more, the products of the oligopeptidases are not amino

to aggregate. Note that this response includes expression of acids, but shorter peptides. Degradation of the peptides is

a nuclear-encoded protein gene termed ubiquitin-like ClpXP. carried out by aminopeptidases that convert the peptides

ClpXP is activated by a homeobox containing the transcrip- into amino acids (Fig. 1). Several cytosolic aminopeptidases

tion factor bZIP, which in turn, is supposed to be activated are known, including leucine aminopeptidase, puromycin-

by a pathway that involves different peptides produced by sensitive aminopeptidase, and bleomycin hydrolase [43,44].

146 e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151

treatment of cells with a proteasome inhibitor (epoxomicin)

4. Identiﬁcation of intracellular peptides

greatly decreases the levels of most intracellular peptides,

thus conﬁrming that the proteasome is the major source of

For a long time it was believed that all peptides generated in

production of these peptides [58]. However, it is not known if

cytosol, nucleus and/or mitochondria were quickly hydrolyzed

the proteasome-mediated generation of the intracellular pep-

to amino acids, and reused in the synthesis of new proteins

tides is part of the degradation pathway of the proteins, or if

and/or metabolic intermediates [2,3]. However, development

this reﬂects a more selective processing step. For example, the

of a “substrate capture assay” that used point mutations

proteasome can perform limited cleavages of proteins such as

for inactivating the catalytic site of oligopeptidases allowed

NF-kappaB [59].

identiﬁcation of several peptides derived from intracellular

proteins in the rat brain [45]. In parallel, the development

of mass spectrometry-based peptidomic techniques made it

5. Intracellular peptides and cell

possible to identify the most abundant peptides present in a communication

tissue, cell line, or other biological sample, without the use of

the substrate capture assay. Initial studies examining extracts Because the intracellular peptides do not generally reﬂect the

of rodent brain identiﬁed hundreds of peptides, most of which degradome of the cell, it is likely that they are produced for a

were subsequently found to represent postmortem degrada- purpose. Furthermore, as described above, synthetic peptides

tion [46]. This postmortem change could be prevented by rapid introduced into cells can perform a variety of functions. There-

heating of the brain tissue, either by microwave irradiation or fore, it is likely that the endogenous intracellular peptides are

by rapid removal of the tissue and heating prior to dissection able to perform the same functions that have been found for

into regions [46–49]. Another problem was the degradation of synthetic peptides (Fig. 2), either outside or inside the cell.

proteins and peptides during the extraction process. Many of Functions outside of the cell include roles as intercellular

the early studies on neuropeptides used hot acid to extract messengers, like classic neuropeptides that are produced in

the peptides but for peptidomics this was found to induce the secretory pathway and secreted upon stimulation. For this

the cleavage of bonds adjacent to aspartate residues, with to occur, the intracellular peptides would need to be secreted

aspartyl–prolyl bonds especially sensitive to hot acid [50]. The from the cell and then interact with another cell in a way that

postmortem and extraction artifacts could be prevented by affected the cell’s function. A precedent for this type of activ-

heat inactivation of the brain tissue followed by extraction ity is yeast a-factor, mentioned above. Intracellular peptides

in ice-cold acid, thus allowing the detection of many neuro- found in mouse brain (and other tissues) that appear to func-

peptides in addition to intracellular peptides [50]. Altogether, tion in cell-cell communication include the hemopressins, a

the use of the substrate capture assay and the development family of related peptides produced from alpha- and beta-

of modern mass spectrometry techniques revealed the pres- hemoglobin. The original hemopressin peptide is a 9-residue

ence of a large number of peptides derived from intracellular peptide fragment of alpha-hemoglobin, which was found to be

proteins that escape further degradation [51]. an antagonist/inverse agonist of the CB1 cannabinoid recep-

Currently, more than 400 intracellular peptides have been tor. N-terminally extended forms of 11 and 12 residues were

identiﬁed in several human cell lines and mouse tissue subsequently identiﬁed and found to have agonistic and nega-

extracts [51–54]. Although some of the precursor proteins of tive allosteric properties on CB1 [60,61] these longer forms may

these peptides are commonly found in the cytosol, mitochon- represent the major forms of hemopressin peptides in brain.

dria and/or nucleus, only a few of them correspond to the most Although many people consider hemoglobin to be a blood cell-

abundant proteins in a cell [53,55]. Moreover, only some of speciﬁc protein, it is expressed in a number of different cell

the protein precursors of intracellular peptides display high types, including neurons and glia [62,63]. The 12-residue form

turnover rates [53,56] or ubiquitin labels [57]. Taken together, of hemopressin was recently found to be secreted from brain

these ﬁndings indicate that the precursor proteins of intracel- slices, supporting the proposed role for this peptide in inter-

lular peptides are not the most rapidly degraded proteins in cellular signaling [64]. A number of other intracellular peptides

the cells. and proteins are known to be secreted, including some inter-

Because the majority of the intracellular peptides detected leukins, growth factors, cytokines, and other molecules [65].

in extracts of human cell lines or mouse tissues are not derived The mechanisms by which this secretion occurs have not

from the most abundant or most unstable proteins, it is pos- been fully elucidated, and this is an important area for further

sible that these peptides are produced from selective cleavage research.

of proteins. In addition to the proteasome, intracellular pro-

teases such as caspases and calpains can execute limited

cleavages of proteins such as hemoglobin. However, the speci- 6. Intracellular peptides and the

ﬁcity of caspases for cleavage sites containing aspartic acid modulation of signal transduction

does not ﬁt with the sequences of the assessed intracellular

peptides since most of the peptides are produced by cleav-

In a recent study of peptides secreted from brain slices, only

ages at hydrophobic residues. Moreover, it is unlikely that

a subset of the peptides detected in the tissue was found

calpain contributes to the production of intracellular pep-

to be secreted from the cells, and the majority of the pep-

tides based on the ﬁnding that activation of this enzyme (by

tides were not secreted [64]. Thus, it is likely that intracellular

treating cells with a calcium ionophore) did not affect the lev-

peptides are functional within the cell. Several possible roles

els of intracellular peptides [53]. Recently, it was shown that

have been proposed. One of these roles isin the modulation

e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151 147

Fig. 2 – Potential functions for endogenous peptides. In all of the examples shown, the peptide is indicated in red. (A)

Peptide binds to receptor, functions as agonist, antagonist, or allosteric modulator. Classical neuropeptides are generally

receptor agonists. Peptides that are produced in the cytosol and secreted can also bind to receptors, as in the case of the

hemopressin peptides, which function as CB1 cannabinoid receptor agonists or antagonists/inverse agonists depending on

the length of the peptide [60]. Intracellular peptides that are not secreted may potentially bind to cysotolic domains on

receptors and alter signaling. (B) Peptide binds to active site or allosteric site of enzyme and alters activity. Examples of

enzymes inhibited by endogenous peptides include proprotein convertase 2 [77], proprotein convertase 1 [78], neprilysin

[79], and insulin-regulated aminopeptidase [80]. In these examples, the peptide binds to the active site and causes the

enzyme to be inactive, as shown in the ﬁgure. Many other enzymes have been found to be inhibited by synthetic peptides,

raising the possibility that intracellular peptides perform this function. It is also possible that a peptide binds to an

allosteric site and activates the enzyme (not shown in ﬁgure). (C) Peptide binds to protein and modiﬁes the ability of the

protein to interact with other proteins. Many proteins form multimers that affect the properties of the protein, and synthetic

peptides have been used to disrupt these interactions and alter the protein’s function [81]. In some cases, the synthetic

peptides activate the protein by mimicking the effect of the protein–protein interaction, in other cases the peptides inhibit

the protein by blocking the protein–protein interaction. It is possible that endogenous peptides perform similar functions as

found for synthetic peptides. (D) Peptide binds to protein and alters intramolecular folding. Many proteins undergo

conformational changes that alter the protein’s function, and synthetic peptides have been used to alter these changes by

binding preferentially to one state of the folded protein [82]. It is possible that endogenous peptides perform similar

functions. (E) Peptide binds to protein and alters its targeting. Synthetic peptides have been used to alter the distribution of

a protein through a combination of the above (altering protein–protein interactions and/or protein folding), or through

another mechanism, such as blocking the interaction of a protein with a membrane or other intracellular molecule [20]. (F):

Peptide binds to protein and blocks its degradation. It is possible that the interaction of a peptide and protein alters the

targeting of the protein to lysosomes or to the proteasome, thereby affecting protein degradation.

of post-translational modiﬁcations. The majority of the pep- be natural competitors of protein phosphorylation [15,66]. In

tides detected using the substrate capture assay described addition, Machado and co-workers demonstrated in vitro that

above have at least one potential site for post-translational phosphorylation of peptides changed their metabolic rates

modiﬁcation, raising the possibility that these peptides could by endopeptidase 24.15 and neurolysin, which should change

148 e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151

their susceptibility to peptidase degradation [67]. Accordingly, back into 3T3-L1 adipocytes [70]. These results support ear-

to test their biological activity three intracellular peptides lier ﬁndings [66] suggesting that intracellular peptides from

were chosen based on the presence of a protein kinase C adipocytes may regulate insulin signal transduction and glu-

(PKC) phosphorylation site and on their binding to the cat- cose uptake [70]. Therefore, intracellular peptides were shown

alytically inactive endopeptidase 24.15 using the substrate to modulate signal transduction of both GPCRs and tyrosine

capture assay. When these peptides were applied externally kinase receptors.

to cells, they were not able to change signaling by G protein-

coupled receptors (GPCRs) but when the same peptides were

attached to a cell-penetrating peptide (CPP), the peptides syn- 7. Potential roles for intracellular peptides

ergistically modulated angiotensin II and isoproterenol signal in the modulation of protein interactions

transduction in HEK293 and CHO cells [68]. Of the three pep-

tides tested, two of them could be phosphorylated by PKC. In addition to a competitive action on protein phosphory-

Considering that all three peptides modiﬁed angiotensin II- lation, a possible general mechanism by which intracellular

induced or isoproterenol-induced luciferase expression in peptides affect signal transduction is through the modulation

the same fashion, it became clear that phosphorylation of of protein interactions (Fig. 2). To search for proteins that inter-

PKC is not the general mechanism responsible for the effect act with intracellular peptides, several peptides that affect

of peptides on the increase in luciferase expression. Even GPCR signal transduction were immobilized and used for afﬁn-

though the mechanism was not known, these studies pro- ity chromatography [68]. Several proteins that interact with

vided the ﬁrst evidence that natural intracellular peptides the intracellular peptides were detected, including dynamin,

␣

identiﬁed using the substrate capture assay could directly 2-adaptin, and 14-3-3; all of these proteins are involved in

␣

affect GPCRs signal transduction from inside the cells. Over- cell signaling [71,72]. 2-adaptin is the major component of

expression of endopeptidase 24.15, which was shown to protein complexes that link clathrin to receptors in coated

change the intracellular peptidome in HEK293 cells, increased vesicles, and for this reason, it is directly involved in GPCRs

the transcription of luciferase in HEK293 and CHO-S cells internalization [72]. Although many other proteins were found

stimulated by GPCR agonists angiotensin II or isoproterenol, to bind to the immobilized peptide afﬁnity columns, it is

supporting the hypothesis that intracellular peptides modu- unlikely that all of them bind directly to these peptides. Sec-

late signal transduction [68]. Recently, it has been shown that ondary interactions of protein are probably responsible for the

cell signaling stimulated by isoproterenol can be enhanced by large number of proteins identiﬁed [68]. Moreover, two pep-

means of siRNA inhibition of endopeptidase 24.15, which in tides that were found to increase glucose uptake by 3T3-L1

parallel altered the intracellular peptidome of HEK293 cells adipocytes were also shown to interact with speciﬁc proteins

[69]. Inhibition of protein kinase A abolished the synergic [70]. One of these proteins, annexin 6, bound only to one of

effect of endopeptidase 24.15 siRNA treatment on isopro- these peptides, whereas heat shock protein 8 was identiﬁed

terenol signaling, further suggesting a role of intracellular as a ligand of both peptides in the extracts from the adi-

peptides in signal transduction [69]. Under this scenario, intra- pose tissues of rats fed with a Western diet but not in rats

cellular oligopeptidases such as endopeptidase 24.15 play an fed the control non-caloric diet [70]. A recent study demon-

important role in regulating the half life of their substrates or strated that peptides FE2 and FE3 among others can modulate

peptide products, with the potential to alter the signal trans- either positively or negatively the formation of protein com-

duction cascade and corresponding cellular functions without plexes related to Ca-calmodulin and 14-3-3 epsilon [73]. The

further energy consumption [38]. peptide VFD7, a proteasome product derived from the S100

Using the substrate capture assay, Heimann et al. demon- calcium-binding protein, is one of the intracellular peptides

␮

strated that high-fat diet-fed mice bearing three functional that efﬁciently (at 1–10 M) inhibited the interaction between

copies of Ace gene produced intracellular peptides of lower Ca -calmodulin and endopeptidase 24.15 [73]. Using isotope

molecular weight, when compared to high-fat diet-fed mice labeling semi-quantitative mass spectrometry it was possible

bearing a single copy of Ace gene [66]. In addition, those to determine the intracellular concentration of VFD7 within

␮

animals containing three copies of the Ace gene showed a HEK293 cells to be in the order of 16 M [73]. When VFD7 was

signiﬁcant improvement in insulin sensitivity when com- added back into the HEK293 cells it enhanced intracellular cal-

pared to the control group. Treatment of mice with losartan cium levels, a similar role of its precursor S100 protein [73].

(angiotensin II receptor antagonist) had no effect on weight This ﬁnding suggests the exciting possibility that intracellu-

gain or on the development of insulin resistance, suggesting lar peptides can modulate speciﬁc protein interactions within

that alternative mechanisms involving intracellular content cells. Further studies are necessary to investigate the in vivo

of peptides may be related to an improvement of insulin effect of intracellular peptides in protein network regulation.

resistance observed in those animals [66]. Semi-quantitative

liquid chromatographic mass spectrometric (LC/MS/MS) anal-

ysis have been used for determination of intracellular peptide 8. Summary and further directions

content in adipose tissue of rats fed with a hypercaloric

western diet [70]. The intracellular levels of two out of ten There are many similarities between intracellular peptides

intracellular peptides, found in the epididymal adipose tissue and microRNA. Generation of microRNA and intracellular pep-

of animals, were at least two times higher in rats fed with the tides both requires enzymatic processing starting from a long

hypercaloric diet when compared to the control group. These molecule to produce smaller ones with the same chemical

two peptides signiﬁcantly increased glucose uptake if added nature that regulate cell function (Fig. 1). The mechanism of

e u p a o p e n p r o t e o m i c s 3 ( 2 0 1 4 ) 143–151 149

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reveal residues critical for processing, activity, and export.

teins can be controlled. Within the cells, intracellular peptides

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The authors gratefully acknowledge support by the

Ferro ES, et al. Thimet oligopeptidase and the stability of

United States National Institutes of Health Grant R01-

MHC class I epitopes in macrophage cytosol. Biochem

DA004494, Brazilian National Research Council (CNPq; Grant

Biophys Res Commun 1999;255:596–601.

559698/2009-7, Rede GENOPROT), Fundac¸ão de Amparo

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