To cleave or not to cleave — neuronal innervation and dementia
The 16-kDa β-nerve growth factor (β-NGF) is the prototypic target-derived neurotrophin
, and biochemical studies show that furin is the principal endoprotease that cleaves pro-β-NGF10,22
(). Surprisingly, the furin-catalysed processing of pro-β-NGF controls whether the neurotrophin activates cell-survival or cell-death pathways within innervating neurons66
. Processed β-NGF mediates cell survival through high-affinity binding to the Trk proto-oncogene receptor tyrosine kinases, which mediate the trophic effects of β-NGF and other neurotrophins. Conversely, secreted, unprocessed pro-β-NGF mediates apoptosis by high-affinity binding to the 75-kDa neurotrophin receptor (p75NTR
). This receptor is a member of the tumour necrosis factor (TNF) receptor/FAS family that antagonizes the trophic signalling mediated by β-NGF and Trk receptors. Regulation of furin activity might therefore have a central role in determining which neurons form synaptic complexes and which neurons die ().
Figure 6 Furin in development, homeostasis and disease. a | Furin-mediated cleavage of pro-β-nerve growth factor (NGF) produces the 13-kDa β-NGF neurotrophin that binds to Trk receptors to promote synaptic innervation. By contrast, inhibition or (more ...)
This control of furin activity might well extend to additional developmental programmes. For example, furin cleavage of the transmembrane receptor Notch is required for the release of the Notch intracellular domain by γ-secretase proteolysis. This intracellular domain then binds to the transcriptional regulator CSL (for ‘C promoter binding factor/Suppressor of Hairless/Lag-1’), which activates genes required for cell–cell communication during development67
. By contrast, uncleaved Notch mediates a distinct signalling pathway that inhibits cell differentiation68
. How furin activity is controlled to regulate the processing of these substrates is unknown.
Furin's role in the α-, β- and γ-secretase-mediated processing of the β-amyloid precursor protein (APP) helps to determine whether APP-derived peptides enhance NGF signalling to innervating neurons or cause the massive neurodegeneration that is associated with Alzheimer's disease69,70
. The extracellular domain of APP is cleaved by α-secretase to produce soluble APPs that enhance the anti-apoptotic and neuroprotective activities of NGF71,72
. By contrast, cleavage of APP by a combination of the β- and γ-secretases releases the amyloidogenic βAPP1–40
and related peptides, which form amyloid plaques and are responsible for the neurodegeneration that is suffered by individuals with Alzheimer's disease73
Neurological abnormality caused by deposits of proteinaceous matter.
Signalling centre in the dental epithelium that controls tooth morphogenesis.
Recent studies point to an essential role for furin in the activation of both α- and β-secretase. Two members of the ADAMs (for ‘a disintegrin and metalloproteinase-like’) family of zinc metalloproteinases — ADAM10 and ADAM17 — have been implicated as the α-secretase74,75
. Characteristic of this protein family, both ADAM10 and ADAM17 contain propeptides that are linked to their catalytic domains by a consensus furin motif, and cleavage at this site is required for their activation76,77
. Interestingly, PC7 might activate the α-secretase under basal conditions, whereas furin might activate it following protein kinase C activation, which increases the α-secretase-catalysed release of soluble APP77
.The β-secretase, also called β-site APP-cleaving enzyme (BACE), is a type-I membrane protein that localizes to the TGN/endosomal system and requires proteolytic removal of its proregion by furin at a minimal furin site (–Arg–Leu–Pro–Arg–↓
. The similarities in BACE and furin trafficking further support the idea that furin is the BACE-activating enzyme81,82
Alzheimer's is only one type of amyloid dementia
in which furin has a crucial role. Two separate mutations in the BRI
gene that encodes a widely expressed type-II membrane protein cause either familial British dementia (FBD) or familial Danish dementia (FDD). In healthy individuals, cleavage of this protein by furin, or possibly PC7, at an atypical furin site, which contains a lysine at P6 (–Lys–Gly–Ile–Gln–Lys–Arg–↓
(where Gln is glutamine)), releases a 23-residue carboxy-terminal peptide with an unidentified function83,84
. However, the nucleotide transversion or decamer duplication in FBD and FDD, respectively, causes aberrant 34-residue amyloidogenic peptides to be produced on furin cleavage83-85
Recent studies also show a role for furin in both Finnish- and Danish-type familial amyloidoses. Both diseases are caused by mutations that disrupt the binding of calcium to plasma gelsolin, which is a circulating scavenger of extracellular actin31,86,87
. The disrupted calcium binding causes aberrant cleavage by furin at the carboxy-terminal side of a cryptic –Arg–Val–Val–Arg–↓
site, which is normally buried in the core of the wild-type molecule. The furin-mediated cleavage initiates the release of a 70-amino-acid amyloidogenic peptide87
Furin, the TNFs, and the TGF-βs — short- versus long-range signalling in development and disease
The proteolytic release of TNF-α from the plasma membrane by ADAM17 has long been regarded as a key mechanism that mediates juxtacrine- versus paracrine-signalling of this cytokine family88
. Recently, however, furin has been shown to control the signalling range of another TNF family member — ectodysplasin-A (Eda-1; ). Eda-1 is a type II plasma membrane protein that controls the formation of several epithelial tissues, including hair, teeth and eccrine sweat glands. The earliest expression of Eda-1 and its receptor — EDAR — is in the partially overlapping regions of the thickened dental epithelium89
. During proliferation of the epithelium into the underlying mesenchyme to form the tooth bud, EDAR expression accompanies the leading edge of the epithelial layer and is ultimately confined to the enamel knot
, whereas Eda-1 remains several cell distances away in the outer epithelium. A furin-mediated switch from juxtacrine to paracrine signalling might accompany the spatial uncoupling of the receptor and ligand (). Mutations in the furin cleavage site of Eda-1 account for ~20% of all know mutations in X-linked hydrohidrotic ectodermal dysplasia, and block the ability of Eda-1 to signal in a paracrine fashion90,91
The importance of furin for the signalling of two other TNF family members — B-cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) — indicates a broad role for furin in controlling TNF function92-94
Furin's role in activating members of the TNF family is surpassed by its role in controlling TGF-β-family signalling. Inactivation of the furin gene in mice creates an embryonic lethal phenotype, with death occurring at an early embryonic stage95
. Furin is required both in the extra-embryonic tissues and in the cardiogenic mesoderm to promote yolk sac vasculogenesis and ventral closure, heart-looping and axial rotation. The failure to maintain asymmetry in the embryo is likely to arise from a block in the furin-catalysed production of the TGF-β family members Nodal and Lefty-2 (ref
). Consistent with this model, furin cleaves several TGF-β members, including TGF- β1 and bone morphogenetic protein-4 (BMP-4)97,98
. Moreover, disrupting pro-BMP-4 maturation in four-cell Xenopus laevis
embryos results in a dorsalized phenotype that mimics the phenotype that is observed when the BMP-4 signalling pathway is disrupted98
Secreted signalling molecules that govern developmental patterns and axis formation by producing a concentration gradient emanating from the cells in which they are synthesized.
Aggressive malignant brain tumours that are derived from astrocytes and that account for ~30% of all primary brain tumours.
Furin's autoactivation method also seems to be used by BMP-4 to control its signalling strength and range during embryogenesis99
. Furin first cleaves pro-BMP-4 at the consensus furin site that joins the pro- and BMP-4 domains (–Arg–Ser–Lys–Arg↓
–), followed by a second cleavage at a minimal consensus furin site within the propeptide (–Arg–Ile–Ser–Arg↓
–). The context of the two sites ensures the ordered processing of pro-BMP-4 and the correct activity of this morphogen
. The presence of consensus and minimal furin sites in other BMP-4-related signalling molecules10,22,99
indicates that the ‘measure once, cut twice’ method is used to control signalling gradients in many organisms. Moreover, this method might extend to viral pathogenesis, in which generation of the correctly folded, respiratory-syncytical-virus (RSV) fusion protein requires sequential cleavage at two furin sites to produce infectious progeny100,101
Although furin-catalysed TGF-β activation is essential for embryogenesis, this pathway causes disease in adults. Furin and TGF-β cooperate in a novel positive feedback loop that exacerbates rheumatoid arthritis (). TGF-β can bind to its own receptor to stimulate furin gene transcription by a SMAD2 and mitogen-activated protein kinase (MAPK) convergent pathway102-104
. In synoviocytes, which are fibroblast- and macrophage-like cells that line the synovium of joints, the amplified levels of furin and TGF-β combine to increase the levels of ADAMTS-4 (a disintegrin and metalloprotease with thrombospondin motifs-4). ADAMTS-4 (previously identified as aggrecanase-1) is a member of a new family of ADAMs proteases, and it degrades the cartilage protein aggrecan and causes rheumatoid arthritis105,106
Furin and tumour metastasis
Furin is upregulated in several cancers, including non-small-cell lung carcinomas, squamous-cell carcinomas of the head and neck, and glioblastomas107
. Moreover, the increased levels of furin in tumours correlate both with the increased aggressiveness of head, neck and lung cancers and with an increase in the levels of one of its substrates — membrane type 1-matrix metalloproteinase (MT1-MMP)108,109
. MT1-MMP activates extracellular pro-MMP2 (pro-gelatinase) to induce rapid tumour growth and neovascularization110
(). Activation of MMPs classically uses a cysteine-switch mechanism, in which the catalytic-site zinc atom that is bound to a cysteine residue in the pro-region of the latent pro-enzyme switches to binding a water molecule in the active protease. However, activation of MT1-MMP and related family members seems decidedly more complex and requires furin-mediated cleavage of their pro-region111,112
. Furin inhibitors, including α1
-PDX, block the activation of MT1-MMP in head, neck and oral squamous-cell carcinomas, which leads to a block in both MMP2 activation and tumour metastasis in transplanted mice109,113
. The fact that the MT1-MMP/MMP2 axis is essential for alveolization
of the embryonic lung114
provides another example of a furin-activated cascade that is essential in embryogenesis but that is detrimental in adults.
A second furin substrate, insulin-like growth factor-1 (IGF1), is upregulated in colon, breast, prostate and lung cancers. Its receptor, IGF1R, which is also a furin substrate, is upregulated on the surface of the tumour cells115
. IGF1 and IGF1R processing are catalysed by furin or PC5/6A, and inhibition of this processing by α1
-PDX reduces the incidence, size and vascularization of tumour development in transplanted mice116
De novo stimulation of new blood supplies to a growing tumour.
Encompasses the latter stages of lung development, which begin with bronchial and respiratory-tree development, and culminate in the formation of terminal saccules and alveoli to facilitate efficient gas exchange.
Furin is not the only PC that is associated with a poor prognosis for many cancers. The furin homologue, PACE4 (; ), is upregulated in breast tumours117
, and expression of this PC increases the invasiveness of mouse squamous-cell carcinomas by converting them to more aggressive, poorly differentiated, spindle-cell carcinomas118
. Together, these studies indicate that inhibiting PCs might be a novel approach to combating various aggressive cancers.
Anthrax, AIDS, Ebola> — what next?
Early studies showing furin's role in both anthrax toxin activation and avian influenza virus HA maturation merely provided a glimpse into the devastating role of furin in the activation of various bacterial and viral pathogens. The analysis of bacterial toxin activation has further illuminated distinct roles for furin-catalysed proprotein processing at the cell surface or early endosomes, providing a single perspective for unravelling the regulation of protein trafficking in mammalian cells. Cell-surface furin activates the anthrax toxin6,119
— a now infamous weapon of bioterrorism120
) — as well as the aerolysin toxin, which is a causative agent in many food-borne illnesses121
, and Clostridium septicum
α-toxin, which causes gas gangrene122
(). Cleavage of each toxin by furin is an obligatory step in making the toxin able to form pores in cell membranes.
The anthrax toxin comprises three proteins: PA, protective antigen, so-called for its ability to educe immune protection against anthrax; and two toxic proteins — lethal factor (LF) or oedema factor (EF)123
. LF is a metalloproteinase that cleaves MAPK kinases, whereas EF is a calmodulin-dependent adenylate cyclase123
. The 83-kDa PA molecule that is secreted from the bacterium binds to the anthrax toxin receptor (ATR)124
, and is then cleaved by cell-surface furin to generate a cell-associated 63-kDa PA and a free 20-kDa PA (). The cell-associated PA molecule heptamerizes, binds to either of the two toxic factors, and is then internalized into early endosomes125
. In the early endosomal acid pH environment, the PA heptamer forms a membrane channel that shuttles the toxic factors into the host-cell cytoplasm, which results in oedema, systemic shock and death (). In the absence of furin, the toxin fails to assemble and is not lethal126
. Moreover, mutation of the furin cleavage site results in a dominant-negative protein that binds to ATR but fails to oligomerize127
. Both proaerolysin and Clostridium septicum
α-toxin bind to glycosylphosphatidylinositol-anchored molecules, and, similar to PA, furin cleavage of both molecules is required for them to form ion-permeable heptameric pores in the host-cell plasma membrane, which leads to cell toxicity128,129
Figure 7 Furin activation of the anthrax toxin. Cleavage of anthrax protective antigen (PA) by furin leads to internalization and activation of lethal factor (LF), which is a zinc metalloproteinase that cleaves mitogen-activated protein kinase (MAPK) kinases, (more ...)
Early endosomal furin activates other bacterial toxins, including Pseudomonas
exotoxin A (PEA), shiga toxin (ST), shiga-like toxin-1 (ST-1) and diphtheria (DT) toxins10
(). Unlike the cell-surface-activated toxins, these toxins are all A/B-type toxins that contain an active domain (A) and a binding domain (B) that are joined by a furin cleavage site129
. Following receptor binding, each toxin is endocytosed into early endosomes, where it is cleaved by furin. Cleavage of ST, ST-1 and PEA by furin requires the acidic pH that is characteristic of early endosomal compartments, whereas cleavage of DT does not10
. As in the cancer models discussed, inhibition of furin activity by the extracellular delivery of α1
-PDX protects cells from PEA and other bacterial toxins13
(F. Jean and G.T., unpublished observations). The sensitivity of cells to PEA is increased in the absence of filamin (), which normally tethers furin to the cell surface, indicating that filamin might control the formation of endosomal furin-processing compartments47
. The crystal structure of PEA shows that exposure of the molecule to an acidic pH unmasks the furin cleavage site, which at least partially explains the requirement for acidic pH-dependent furin processing130
Surprisingly, furin cleavage enables PEA, ST/ST-1 and DT to translocate to the cytosol through three distinct trafficking pathways. Following cleavage, the DT B domain forms a channel in the early endosomal membrane that shuttles the A fragment into the host-cell cytosol131
. By contrast, cytosol delivery of both PEA and ST/ST-1 requires retrograde trafficking to the ER, where the toxins are apparently translocated to the cytosol through the sec
. The retrograde trafficking of PEA requires the kdel receptor
, which binds to the processed PEA and retrieves the toxin to the ER132,133
,whereas cleaved ST/ST-1 traffics to the ER through a pathway that is both KDEL-receptor- and copi
-independent, which indicates that vesicle coats other than COPI direct their retrieval to the ER. However, retrieval of ST/ST-1 is dependent on RAB6, a small GTPase that controls intra-Golgi transport and the cycling of Golgi-resident glycosyltransferases through the ER132-134
. As it has also been suggested that furin might be sorted to the ER to metabolize misfolded insulin receptors, it will be important to determine whether furin is also sorted through the ST/ST-1 pathway135
The main protein that forms the pore in the endoplasmic reticulum (ER) membrane that facilitates the translocation of nascently synthesized proteins into the secretory pathway. The SEC61 channel might also be a conduit for the reverse translocation (dislocation) of proteins from the ER into the cytoplasm.
Golgi-localized membrane protein that binds to carboxy-terminal KDEL (–Lys–Asp–Glu–Leu–) motifs, which are present on many resident endoplasmic reticulum (ER) proteins that escape to the Golgi, and that retrieves them to the ER.
(Coatomer protein complex I). A specific type of coat on vesicles that traffic principally between Golgi cisternae and from the Golgi to the endoplasmic reticulum. Also reported on early endosomes.
The extent or degree to which a pathogen can cause disease.
The broad role of furin in activating bacterial toxins is exceeded by its role in activating numerous pathogenic viruses. Many pathogenic viruses, including avian influenza virus, HIV-1, measles virus and RSV, express envelope glycoproteins that must be cleaved at consensus furin sites to form the mature and fusogenic envelope glycoprotein10,22
. For example, processing of HIV-1 gp160 unveils the amino-terminal gp41 fusogenic peptide that is contained within the trimeric gp120/g41 envelope complex. Whether furin or one of the other PCs (for example, PACE4, PC5/6B or PC7) is the in vivo
gp160 convertase is unknown136
. Lovo cells, which lack furin, process HIV-1 gp160 (ref
). Nonetheless, furin inhibitors block processing of HIV-1 gp160 and, in turn, the production of infectious HIV-1, as well as blocking other viruses that require processing of their envelope glycoproteins at consensus furin sites10
. Furin cleaves HIV-1 gp160 at the carboxy-terminal side of the consensus sequence –Arg–Glu–Lys–Arg–↓
. The P3 glutamate in this cleavage site reduces the efficiency of furin processing, and the conservation of this residue in several HIV isolates has raised doubts about furin's involvement in gp160 processing136
. Indeed, mutation of this cleavage site to make a site containing all basic amino acids (–Arg–Arg–Lys–Arg–↓
) enhances processing by furin138
. Surprisingly, however, recombinant HIV containing this all-basic site is attenuated, which indicates a selective advantage for the inefficiently cleaved –Arg–Glu–Lys–Arg–↓
site in HIV-1.
HIV-1 seems to maintain a selective growth advantage by using a suboptimal furin site in its envelope glycoprotein, whereas the analysis of viral tropism — that is, the molecular determinants that enable a virus to spread throughout the body — shows that the virulence
of many deadly viruses (including avian influenza virus, Newcastle Diseases virus and, potentially, Ebola virus) is directly correlated with the ability of these viruses to incorporate a consensus furin cleavage site within their envelope proteins139-142
. For example, the pathogenicity of avian influenza viruses has long been recognized to correlate directly with the cleavability of its fusion protein precursor HA0
, which is cut by furin to generate the fusion-competent HA1
complex. Similar to HIV-1 gp160, cleavage of HA0
exposes the fusogenic peptide located at the amino terminus of HA2
, which can fuse with target-cell membranes.
Avirulent avian influenza viruses, which lack a consensus furin site in HA0
, cause a localized infection in the intestinal tract. However, mutation of the HA0
cleavage site to a consensus furin site enables the virus to be activated by the ubiquitously expressed furin, invariably enabling the virus to spread systemically throughout the bird, including infection of the central nervous system139
. This ability relates to the deadly flu outbreak in Hong Kong in 1997 (BOX 2
). Analysis of the H5N1 influenza virus, which killed at least six people, showed that just two mutations were required to generate the lethal virus — a mutation in a subunit of the viral RNA polymerase PB2, together with the generation of a tandem furin site in the cleavage junction between HA1
. Exactly how the tandem furin site and the PB2 mutation contribute to the increased virulence of influenza H5N1 is unknown. Fortunately, the attenuated infectivity
of this virus, which is also poorly understood, impeded its spread through the population. Nonetheless, the propensity for the rapid mutation and reassortment rate in avian influenza and its documented ability to jump directly from birds to humans underscore our vulnerability to this pathogen144
The importance of furin for pathogen virulence extends to other viruses, including Ebola virus. For example, the highly pathogenic Ebola Zaire and Ivory Coast strains — which cause a massive and sudden (fulminant) haemorrhagic fever that is characterized by massive internal and external bleeding and that kills 90% of the people who contract it — contain a consensus furin site in their envelope glycoprotein (GP)145
. But, by contrast, GP of the relatively milder Ebola Reston strain lacks a consensus furin site. This isolate is not pathogenic to humans141
. Surprisingly, however, despite the apparent underlying structural similarities between HIV-1 gp160, influenza virus HA and Ebola virus GP, furin-catalysed cleavage of GP is not required for membrane fusion in cell-culture models146
. The lack of a requirement for GP processing for membrane fusion is consistent with the presence of an internal fusion sequence in Ebola GP and related flaviviruses147
. What, then, might account for the furin-dependent tropism of Ebola virus? One clue lies in the severe cytotoxicity and marked increase in vascular permeability of GP from the highly pathogenic Ebola Zaire, but not from the apathogenic Reston isolate148,149
. Interestingly, the crucial region of GP required for this toxicity is adjacent to the furin cleavage site, indicating that proteolysis might unveil the cytotoxic domain.
The ability of a pathogen to invade a host and replicate, irrespective of its ability to cause disease.