Protein moonlighting a.k.a. gene sharing has been defined as the concept that one protein processes multiple tasks and plays multiple roles [1]. Thus, multifunctional proteins are designated as moonlighting proteins. Matrix metalloproteinases play multiple roles in extracellular proteolysis and intracellular gene regulation [2,3], prompting us to propose a new definition of Moonlighting Metalloproteinase (MMP).
In this encyclopedia, the followings are reviewed: 1. Structures of MMPs; 2. Extracellular roles of MMPs; 3. "Intracellular roles" of MMPs; 4. "Extracellular vesicle"-associated MMPs; 5. Roles of MMPs in cancers.
1. Structures of MMPs [1]
The matrix metalloproteinase family consists of
The matrix metalloproteinase family consists of
about 30 members that share similarities in their structure, regulation, and function [2]. Earlier studies showed MMPs constitute a large family of zinc/calcium-dependent endopeptidases. All MMPs have principal domains, including (1) A
that share similarities in their structure, regulation, and function [4]. Earlier studies showed MMPs constitute a large family of zinc/calcium-dependent endopeptidases. All MMPs have principal domains, including (1) A
signal peptide (SP) sequenc
e found at the very N-terminus of all MMPs, (2) a
pro-domain
that functions as an intramolecular inhibitor to maintain the enzyme in an inactive state, (3) a
metalloproteinase catalytic domain
that can exert the proteolytic activity, (4) A
linker sequence
connecting the catalytic domains with a following domain, and (5) a
hemopexin-like repeat (PEX or HPX) domain
, which interacts with other molecules and determines the substrate specificity. The PEX domain is present in all MMPs except for MMP-7, -23, and -26.
Proline residues in the middle of the SP can structurally weaken the secretory activities of MMPs [3][4] and thus generate intracellular MMPs. Besides, human MMP-3 contains six
Proline residues in the middle of the SP can structurally weaken the secretory activities of MMPs [2,5] and thus generate intracellular MMPs. Besides, human MMP-3 contains six
nuclear localization signals (NLS)
composed of basic amino acid clusters [5]. MMP3 thus has both extracellular and nuclear functions.
Additionally, gelatinases (MMP-2, -9) contain the
fibronectin type II inserts
in the middle of the catalytic domain. Membrane type (MT) -MMPs contain
type I transmembrane (TM) domains
followed by cytoplasmic tails at the C-terminus. MT-MMPs are composed of MMP-14, -15, -16, and -24. Only MMP-23 contains a cysteine array region and an IgG-like domain.
2. Extracellular roles of MMPs
MMPs cleave substrate proteins in the extracellular space. MMP-dependent proteolysis of
MMPs cleave substrate proteins in the extracellular space. MMP-dependent proteolysis of
extracellular matrix (ECM)
and
intercellular adhesion molecules
enable cells to migrate and invade tissue
microenvironment
. Proteolysis of ECM also triggers the
release of cytokines, chemokines, and growth factors
that activate their
receptors and intracellular signaling pathways. In addition, MMPs also directly cleave and alter activities of growth factors, cytokines, chemokines, and their receptors. For example, MMPs can alter the activity of connective tissue growth factor (CTGF, recently known as cellular communication network factor 2 (CCN2)) by direct cleavage [5]. MMP cleavage of CCNs alters the angiogenic activities of CCNs and VEGF. A disintegrin and metalloproteinases (ADAMs) family members as well as MMPs cleave membrane-bound heparin-binding EGF-like growth factor (HB-EGF) and then release soluble HB-EGF, which stimulates EGFR/ERBB signaling [6].
and intracellular signaling pathways. In addition, MMPs also directly cleave and alter activities of growth factors, cytokines, chemokines, and their receptors. For example, MMPs can alter the activity of connective tissue growth factor (CTGF, recently known as cellular communication network factor 2 (CCN2)) by direct cleavage [6]. MMP cleavage of CCNs alters the angiogenic activities of CCNs and VEGF. A disintegrin and metalloproteinases (ADAMs) family members as well as MMPs cleave membrane-bound heparin-binding EGF-like growth factor (HB-EGF) and then release soluble HB-EGF, which stimulates EGFR/ERBB signaling [7].
Extracellular MMPs are known to be components of the
Extracellular MMPs are known to be components of the
senescence-associated secretory phenotype (SASP) that includes interleukins (IL-1β, IL-1α, IL-6, IL-8) and chemokines (CCL2) as well [7].
that includes interleukins (IL-1β, IL-1α, IL-6, IL-8) and chemokines (CCL2) as well [8].
3. Intracellular roles of MMPs
Intracellular and intranuclear roles for MMPs have also been discovered. MMP-3 possesses several
NLSs
and can translocate into cellular nuclei, at which site MMP-3 can
bind to DNA and chromatin proteins
leading to
transcriptional regulation
of
CTGF/CCN2 gene [3][8]. Promoter analysis of the
gene [2,3]. Promoter analysis of the
CCN2/CTGF
gene revealed a
cis-element, designated transcriptional enhancer dominant in chondrocytes (TRENDIC) [3][9]. One of the TRENDIC-binding proteins was identified to be MMP-3 [3]. MMP-3 overexpression enhanced
-element, designated transcriptional enhancer dominant in chondrocytes (TRENDIC) [2,9]. One of the TRENDIC-binding proteins was identified to be MMP-3 [2]. MMP-3 overexpression enhanced
CCN2/CTGF promoter activity in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 and non-basal type, triple-negative breast cancer cell line MDA-MB-231 [3].
promoter activity in human chondrosarcoma-derived chondrocytic cell line HCS-2/8 and non-basal type, triple-negative breast cancer cell line MDA-MB-231 [2].
Intranuclear translocation of recombinant MMP-3, as well as endogenous MMP-3, were observed under confocal laser scanning microscopy (CLSM) [3].
of recombinant MMP-3, as well as endogenous MMP-3, were observed under confocal laser scanning microscopy (CLSM) [2].
DNA-binding
of MMP-3 was demonstrated by gel shift and chromatin immunoprecipitation (ChIP) assays. An MMP-3 specific inhibitor inhibited the activity of the
CCN2/CTGF promoter [3], suggesting that MMP-3 proteolytic activity was partly involved in the transcriptional role for this enzyme, although the general MMP inhibitor GM6001 or an MMP2/9 inhibitor were each ineffective in this regard [3]. MMP-3 was strongly immunostained in cell nuclei in
promoter [2], suggesting that MMP-3 proteolytic activity was partly involved in the transcriptional role for this enzyme, although the general MMP inhibitor GM6001 or an MMP2/9 inhibitor were each ineffective in this regard [2]. MMP-3 was strongly immunostained in cell nuclei in
cartilage tissues in the normal and arthritic mouse model [3]. These findings demonstrated the role of MMP-3 in
[2]. These findings demonstrated the role of MMP-3 in
gene regulation
.
MMPs also undergo endogenous
MMPs also undergo endogenous
auto-cleavage
MMPs themselves to generate fragments containing the PEX domain. The
PEX domain
transcriptionally activates some members of
heat shock protein (HSP)
genes [10], a process that can contribute to anti-apoptosis and drug resistance. MMP-3 directly interacts with
heterochromatin proteins (HP1)
, members of the
chromobox protein (CBX) family that involve transcriptional and chromosomal control [3][4][10].
family that involve transcriptional and chromosomal control [2,5,10].
MMPs also impact
MMPs also impact
oxidative stress, DNA damage
, and
chromosome instability
in cell nuclei. Intranuclear activities of MMP-2 and MMP-9 were shown to cleave PARP-1 and XRCC1, nuclear matrix proteins, promoting
oxidative DNA damage
, and apoptosis in an ischemic injury model [11].
4. Extracellular vesicle-associated MMPs
Members of the MMP family are often associated with
Members of the MMP family are often associated with
extracellular vesicles (EVs)
[12]. MMP3-rich EVs enhance cancer cell migration and invasion, molecular transmission, and gene activation while
CRISPR/Cas9-based knockout of MMP3 reduced these pro-tumorigenic roles of EVs [8][13].
-based knockout of MMP3 reduced these pro-tumorigenic roles of EVs [3,13].
EV-associated MMP3
was transmissive into recipient cell nuclei, trans-activated the
CCN2/CTGF promoter, and induced CCN2/CTGF production in vitro [8].
promoter, and induced CCN2/CTGF production in vitro [3].
The CRISPR/Cas9-mediated knockout of
The CRISPR/Cas9-mediated knockout of
Mmp3
gene significantly
reduced 3D-tumoroid formation
in vitro, reduced the levels of tetraspanins (CD9 and CD63) in EVs, and resulted in
destabilizing EV structural integrity
[13]. Indeed, the
Mmp3
gene loss was associated with abnormal, disorganized shapes of EVs such as crescent moon-like and broken EVs [13]. MMP3-enriched EVs were highly
penetrative
and transferred deeply into the recipient MMP3-null tumoroids [13]. The addition of MMP3-rich EVs fostered the tumorigenicity and increased the proliferation of MMP3-null cells as judged by the highly significant increase in Ki-67 expression index. Thus, MMP3-rich EVs were highly
transmissive and pro-tumorigenic
in vitro [13].
5. Roles of MMPs in cancers
MMPs represent the most prominent family of proteinases involved in
MMPs represent the most prominent family of proteinases involved in
tumor progression
and are regulators of the
tumor microenvironment [14][15]. MMPs have also been reported to be potent biomarkers of tumor progression as well constituting some of the causal factors that promote multiple processes of tumorigenesis, including oxidative stress-dependent DNA damage and chromosomal instability,
[14,15]. MMPs have also been reported to be potent biomarkers of tumor progression as well constituting some of the causal factors that promote multiple processes of tumorigenesis, including oxidative stress-dependent DNA damage and chromosomal instability,
epithelial-to-mesenchymal transition (EMT)
[16], migration and invasion of cancer cells [17],
angiogenesis
, and
metastasis [14][15][18]. MMP3-induced EMT and genomic instability are mediated by the
[14,15,18]. MMP3-induced EMT and genomic instability are mediated by the
small GTPase Rac1b
and a
reactive oxygen species (ROS) in breast adenocarcinoma and pancreatic cancer [16][19], indicating potent roles for MMPs in proteotoxic and genotoxic stress.
in breast adenocarcinoma and pancreatic cancer [16,19], indicating potent roles for MMPs in proteotoxic and genotoxic stress.
MMPs appear to be appropriate target molecules in treatments of aggressive types of cancers. Although more than 50 types of MMP inhibitors have been investigated in clinical trials for various cancers, all of those trials have so far failed [18]. The involvement of
MMPs appear to be appropriate target molecules in treatments of aggressive types of cancers. Although more than 50 types of MMP inhibitors have been investigated in clinical trials for various cancers, all of those trials have so far failed [18]. The involvement of
intracellular and non-proteolytic roles for MMPs
in cancer has, however, not been well-investigated yet.
High expression of
High expression of
MMP3
mRNA was
prognostically unfavorable in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers [8]. The
in particular types of cancers including head and neck, lung, pancreatic, cervical, stomach, and urothelial cancers [3]. The
RNA interference (RNAi)
-mediated knockdown of MMP-3 and MMP-9
significantly inhibited tumor growth and metastasis
in the tumor allograft mouse model [20].
CRISPR/Cas9-based knockout of MMP3 revealed that MMP3 is essential for the integrities of tumors and their EVs [13].
-based knockout of MMP3 revealed that MMP3 is essential for the integrities of tumors and their EVs [13].
6. References