cullin proteins are molecular scaffolds that have crucial roles in the post - translational modification of cellular proteins involving ubiquitin .
the mammalian cullin protein family comprises eight members ( cul1 to cul7 and parc ) , which are characterized by a cullin homology domain .
cul1 to cul7 assemble multi - subunit cullin - ring e3 ubiquitin ligase ( crl ) complexes , the largest family of e3 ligases with more than 200 members .
although cul7 and parc are present only in chordates , other members of the cullin protein family are found in drosophila melanogaster , caenorhabditis elegans , arabidopsis thaliana and yeast .
a cullin protein tethers both a substrate - targeting unit , often through an adaptor protein , and the ring finger component in a crl .
the cullin - organized crl thus positions a substrate close to the ring - bound e2 ubiquitin - conjugating enzyme , which catalyzes the transfer of ubiquitin to the substrate .
in addition , conjugation of cullins with the ubiquitin - like molecule nedd8 modulates activation of the corresponding crl complex , probably through conformational regulation of the interactions between cullin 's carboxy - terminal tail and crl 's ring subunit . genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective crls .
crls target numerous substrates and thus have an impact on a range of biological processes , including cell growth , development , signal transduction , transcriptional control , genomic integrity and tumor suppression .
moreover , mutations in cul7 and cul4b genes have been linked to hereditary human diseases .
cullins , containing an evolutionarily conserved cullin homology domain , are a family of structurally related proteins required for ubiquitin - dependent protein degradation ( box 1 ) .
kipreos et al .   identified cullins as a novel gene family involved in cell cycle regulation in nematodes .
isolated cdc53 ( cell division control protein 53 ) , a cullin homolog in budding yeast , and elucidated its role in ubiquitin - dependent proteolysis of cell - cycle regulators .
thus , cullins are named after their role in ' culling ' , sorting or ' selecting ' cellular proteins for ubiquitin - mediated proteasomal degradation ( e kipreos , personal communication ) . the cullin gene family is evolutionarily conserved .
table 1 presents a list of the cullin family genes from a range of representative species with respect to their gene organization and expression .
there are seven cullins in mammals ( cul1 to cul3 , cul4a , cul4b , cul5 , cul7 and the closely related p53-associated parkin - like cytoplasmic protein ( parc ) in homo sapiens , mus musculus and rattus norvegicus ) , six in c. elegans ( cul-1 to cul-6 ) and five in drosophila ( cul1 to cul5 ) .
arabidopsis has five cullins ( cul1 , cul2 , cul3a , cul4 and cul5 ) , and yeast genomes encode three cullin proteins ( cul1 , cul3 , cul8 in saccharomyces cerevisiae ; cul1 , cul3 and cul4 in schizosaccharomyces pombe ) . in total ,
490 cullin domains in 490 proteins are described in the smart nrdb database , which estimates 60% conservation in the cullin homology domains .
chromosomal localization of the cullin genes from several representative species gene ids are as listed in entrez gene .
the intron number and transcript information are indicated for the cullin genes in human and mouse . only the variants in entrez gene
the presence of the cul1 to cul5 genes in the early - branching metazoans trichoplax adhaerens and nematostella vectensis indicates that cullin genes are ancient and originated before the separation of the different animal lineages .
an extensive genome - wide analysis of the cullin family has suggested that three ancestral cullin genes , termed ' cul ' , ' cul ' and ' cul ' , appeared in early eukaryotic evolution , from which the cullin genes evolved after the split of the unikonts ( which include animals and fungi ) and the bikonts ( which include plants ) . in this model ,
the human cul1 , cul2 , cul5 , cul7 and parc genes were derived from one common ancestral gene ( cul ) , whereas the cul3 and cul4a/4b genes evolved from two distinct ancestors , the cul and cul gene , respectively .
notably , cul7 and parc , found only in chordates , are highly similar in sequence and both contain a cph ( conserved in cul7 , parc and herc2 ) domain and a doc domain ( similar to the doc1 of the anaphase - promoting complex / cyclosome ) of unknown functions ( figure 2 ) . in human and mouse , cul7 and parc are located on the same chromosome in close proximity ( 260 kb apart ) .
based on these findings , marin et al .   suggested that parc originated from a gene fusion of a duplicate of cul7 and an ariadne gene , which encodes a putative e3 ubiquitin ligase sharing structural similarity with parkin
fungal species contain only three cullin genes : cdc53 , cul3 and cul8 ( also known as rtt101 ) in s. cerevisiae and cul1 , cul3 and cul4 in s. pombe .
however , cul8/rtt101 in saccharomycotina differs significantly from the cul4-like genes of other fungi ( such as ascomycetes and basidiomycetes ) .
it was postulated that cul8/rtt101 originated from the cul4 gene that underwent an accelerated evolution , or that saccharomycotina has lost their cul4-like gene and the cul8/rtt101 gene arose in parallel as a result of gene duplication .
phylogenetic tree based on the alignment of cullins of homo sapiens ( hs ) , mus musculus ( mm ) , rattus norvegicus ( rn ) , drosophila melanogaster ( dm ) and caenorhabditis elegans ( ce ) . gene ids are as listed in table 1 .
the bar indicates the proportion of amino acid sites at which two compared sequences are different .
cullin repeat 1 ( cr1 ) anchors the cognate adaptor proteins , and the cullin homology domain ( ch ) at the carboxyl terminus is critical for binding of the ring - finger protein .
size and location of the individual domains are schematic representations and do not depict the exact proportions .
abbreviations : aa , amino acids ; ch , cullin homology domain ; cph , conserved domain in cul7 , parc and herc2 ; cr , cullin repeat ; doc , a domain similar to the doc1 domain of the anaphase - promoting complex / cyclosome but of unknown function ; ibr , in between ring ; ring , really interesting new gene .
ring ( really interesting new gene)-type e3 ubiquitin ligases orchestrate ubiquitination by simultaneously binding to a protein substrate and anchoring an e2 ubiquitin - conjugating enzyme through the ring domain ( for detailed description of the actions of e2 and e3 in ubiquitination , see boxes 1 and 2 and figure 3 ) .
cullins are molecular scaffolds that organize the largest class of ring e3 ligases , known as the cullin - ring ligase complexes ( crls ) . below
the ubiquitin - proteasome system . the conserved 76-amino - acid polypeptide ubiquitin ( ub )
is activated in an atp - dependent reaction by an e1 ubiquitin - activating enzyme and transferred to an e2 ubiquitin - conjugating enzyme .
an e3 ubiquitin ligase binds both the substrate protein and a ubiquitin - charged e2 enzyme for ubiquitin transfer , resulting in the mono- , multi- ( not shown ) or polyubiquitination of the substrate .
the mode of ubiquitination determines whether the substrate protein is degraded by the 26s proteasome or altered in a non - proteolytic manner .
the structural properties of cullins have been revealed in the context of crls by high - resolution structural studies and biochemical reconstitution experiments ( table 2 ) .
although all the cullin ( and crl ) structures that have been solved so far have been mammalian proteins ( table 2 ) , the interactions between cullins and their crl components have been analyzed using biochemical assays in systems from yeast to humans , highlighting an extraordinary conservation regarding the scaffolding functions of cullins .
thus , the cullin and crl structural models discussed in this section are generally applicable to counterparts of all origins .
cullin structures and cullin - ring e3 complex assembly * crystal structures of crl complexes were solved with recombinant human proteins .
cul1 to cul5 have a long stalk - like amino - terminal domain ( ntd ) , consisting of three cullin repeats ( cr1 to cr3 ) , and a globular carboxy - terminal domain ( ctd ) , which harbors a signature cullin homology domain ( ch ) , a highly conserved stretch of about 200 amino acids ( figure 2 ) .
the cullin ctd binds to its ring partner , regulator of cullins 1 ( roc1 ) or roc2 ( also called ring box protein ( rbx)1 and rbx2 , respectively ) , which recruits the ubiquitin - loaded e2 enzymes for catalysis .
the cul1-roc1 association is established by multiple interface interactions , primarily involving cul1 's / domain and the amino - terminal s1 -strand of roc1 , which enable the formation of an intermolecular / hydrophobic core that essentially renders cul1-roc1 physically inseparable .
the cullin - ring interaction creates a catalytic core and is the most characteristic structural feature that defines crls . on the basis of structural studies of the human s - phase kinase - associated protein 1 ( skp1)-cul1-f - box ( scf ) complex   and the cul4a ring ( crl4a ) complex ,
cullins organize crls by forming two distinct modules : a substrate - targeting unit , composed of a substrate - recognition protein and an adaptor protein that links the module to the cullin , and the ring component that is active in recruiting an e2 ubiquitin - conjugating enzyme ( figure 4 ) .
for instance , scf contains a substrate recognition subunit known as the f - box protein , which is characterized by a 40-amino - acid f - box domain .
the skp1 adaptor protein mediates binding of the f - box protein to the amino terminus of cul1 . in crl4a , the substrate - targeting unit
is composed of damage - specific dna binding protein 1 ( ddb1 ) as the adaptor protein , and a member of the ddb1 and cul4 associated factor ( dcaf ) family that recognizes a substrate .
instead , it incorporates btb ( bric - a - brac , tramtrack , broad - complex ) , a dual function molecule capable of binding to cul3 and targeting a substrate ( figure 4 ) .
( scf , also called crl1 , is historically the prototype of all crls and thus the name scf remains commonly used in the current literature . )
cullin proteins are molecular scaffolds that assemble multi - subunit cullin - ring e3 ubiquitin ligase ( crl ) complexes .
the mammalian cullin protein family comprises eight members ( cul1 to cul7 and parc ) . in crl ,
a cullin protein tethers both a substrate - recognition subunit , often through an adaptor protein , and the ring finger component .
the cullin - organized crl thus positions a substrate in close proximity to the ring - bound e2 ubiquitin - conjugating enzyme ( not shown ) , which catalyzes the transfer of ubiquitin to the substrate .
btb , bric - a - brac , tramtrack , broad - complex domain ; dcaf , ddb1-cul4 associated factor ; ddb1 , dna damage - binding protein 1 ; fbw8 , f - box and wd repeat domain containing protein 8 ; parc , p53-associated parkin - like cytoplasmic protein ; socs , suppressors of cytokine signaling ; skp1 , s - phase kinase - associated protein 1 ; vhl - box , von hippel - lindau box .
accumulating evidence points to a common mechanism by which cul1 to cul5 build a substrate - targeting unit .
cul1 to cul5 use amino - terminal helices h2 and h5 of cr1 to anchor their cognate adaptors .
there are two distinct types of recognition fold in the adaptor ( table 2 ) . in the scf , crl2 , crl3 and crl5 e3s , different adaptors
( skp1 , elongin c ( eloc ) or btb ) share a similar structural motif termed the skp1/btb / pox virus and zinc finger ( poz ) fold , which is a primary determinant for affinity interactions with the cullin amino terminus .
by contrast , the ddb1 adaptor of crl4 lacks the skp1/btb / poz fold and instead uses its bpb domain to interact with the cul4a h2 and h5 helices , as well as the amino - terminal extension . in this regard ,
it is worth commenting on the enormous structural complexity of ddb1 , a 127 kda protein containing three large propeller folds , which potentially enable multiple interactions with cellular proteins .
indeed , ddb1 was found to form a complex with de - etiolated 1 ( det1 ) , ddb1 associated 1 ( dda1 ) and the e2 ubiquitin - conjugating enzyme ube2e .
initial efforts to isolate the human cul4a - containing complexes resulted in very large complexes that contained the constitutive photomorphogenesis 9 ( cop9 ) signalosome .
the role of the cop9 signalosome in crl function is discussed in the next section .
structural and biochemical analyses have revealed additional protein - protein interactions that contribute to the cullin - mediated crl assembly .
in addition to the skp1-cul1 interactions , skp2 's f - box domain also binds to cul1 , thus contributing to the assembly of the scfcomplex .
although cul3 mediates interactions to btb proteins through the skp1/btb / poz fold , it binds to a conserved helical structure carboxy - terminal of the btb domain , which was named ' 3-box ' for cul3-interacting box   ( table 2 ) .
the cul3 - 3-box association strengthens the cul3-btb protein interactions . despite sharing the identical adaptor protein elongin c ( eloc ) , cul2 and cul5 direct the assembly of distinct e3 complexes : crl2 with von hippel - lindau ( vhl ) or related bc box proteins , and crl5 containing suppressors of cytokine signaling ( socs)-box proteins ( figure 4 ) .
however , it is unclear whether cul2 and cul5 recognize specific determinants within vhl and socs - box proteins , respectively .
a recent study provided some insight into how cul5 assembles into a crl5 complex with the hiv protein virion infectivity factor ( vif ; a socs - box protein ) , thereby yielding an e3 ligase that targets the human antiviral protein apobec3 g for proteasomal degradation .
it seems that loops 2 and 5 of cul5 are engaged in interactions with vif 's socs - box and zinc finger motif ( h-(x)5-c-(x)17 - 18-c-(x)3 - 5-h ) , respectively . in summary
, it seems that to assemble crls , cullins not only bind to a common recognition fold in the adaptor , such as the skp1/btb / poz motif ( table 2 ) , but also form interface interactions with structural determinants within the substrate - recognition molecules ( srms ) that include f - box , btb , vhl and socs proteins . however , future structural and biochemical studies , using a larger set of substrates , are required to more rigorously define the ' srm determinants ' . to understand the differential ability of cul2 and cul5 to assemble crl2 and crl5 , respectively , it is critical to solve their structures , especially the amino terminus .
there are no structures available that reveal the three - dimensional organization of either cul7 or parc .
although cul7 resembles cul1 in using the skp1 adaptor , it remains to be determined how cul7 selects f - box and wd - repeat - domain - containing protein 8 ( fbw8 ) . at present , it is also unclear whether parc forms a multi - subunit complex .
cullin family proteins are involved in a diverse array of functions , including cell - cycle control , dna replication and development .
the major physiological functions of the cullin family proteins have been revealed by genetic ablation experiments in a variety of metazoan model organisms , including mouse , c. elegans and drosophila ( table 3 ) . in arabidopsis ,
crls regulate hormonal signaling , light responses , circadian rhythms and photomorphogenesis ( for a recent review , see ) .
the cullin family proteins seem to be widely expressed and to locate both to the nucleus and cytoplasm , but there are no compelling data suggesting that cullin activity is controlled by subcellular localization or by differential expression in a tissue - specific manner .
major physiological roles of cullins revealed by deletion studies with model organisms e , embryonic day ; ko , knockout .
cul1 and cul3 mouse knockout experiments have revealed their indispensable roles in cell cycle progression and early embryogenesis ( table 3 ) .
it was the pioneering work using the budding and fission yeast systems that led to the discovery of cullins and other crl components and their role in cell cycle control ( reviewed in ) .
work in c. elegans and drosophila has demonstrated the requirement of cul1 for cell cycle progression ( table 3 ) .
mechanistically , it is believed that the cul1-based scf regulates the mammalian cell cycle , at least in part , by using the skp2 f - box protein , which directs the ubiquitin - dependent degradation of p27 and p21 ( inhibitors of cyclin - dependent kinases ) , thereby activating cyclin - dependent kinases .
scf malfunction has been linked to malignancy , as mutations in the fbw7 f - box protein are frequently found in a variety of human cancers .
studies with cul4 deletion in c. elegans have established a crucial role for cul4 in dna replication ( table 3 ) .
it is well accepted that the crl4 complex with the dcaf protein cdt2 as substrate - recognition molecule ( referred to as crl4 ) targets the replication initiation factor cdt1 for degradation , thereby preventing dna re - replication . in mammals , cul4a and cul4b
are believed to be functionally redundant , as deletion of cul4a in mice resulted in viable animals and relatively subtle phenotypes ( table 3 ) .
clearly , complete understanding of the physiological functions of cul4 in mouse development requires future studies with animals lacking cul4b and cul4a / cul4b .
. however , crl2has a critical role in control of oxygen homeostasis , acting by targeting the  subunit of hypoxia - inducing transcription factor ( hif ) for degradation ( reviewed in ) .
tissue - specific gene targeting of vhl in mice has demonstrated that efficient execution of crl2-mediated hif-1 proteolysis under normal levels of oxygen is fundamentally important for survival , proliferation , differentiation and normal physiology of many cell types ( reviewed in ) .
these studies have explained the tumor suppressor function of vhl , whose germline mutations inactivate its ability to form the crl2 complex or bind to hif- , leading to the formation of highly vascularized tumors such as renal clear - cell carcinomas .
two hereditary human diseases have been linked to genes encoding members of the cullin protein family .
mutations in the cul7 gene were linked to 3-m syndrome ( online mendelian inheritance in man ( omim ) i d 273750 ) , an autosomal - recessive disorder characterized by pre- and postnatal growth retardation ( final height 3 to 4 standard deviations below the mean for the population ) , facial dysmorphism , large head circumference , normal intelligence , and skeletal anomalies that include long slender tubular bones and tall vertebral bodies .
identified cul7 gene mutations in 52 out of 62 cases ( 84% ) , arguing for cul7 as the major disease gene of 3-m syndrome .
the mutations were located throughout the cul7 gene and most are predicted to cause premature termination of translation .
reverse transcriptase ( rt)-pcr analysis of patient fibroblast mrna detected a cul7-specific transcript , but at reduced levels , arguing that cul7 mrna is expressed at least in a subset of 3-m syndrome patients .
approximately 50% of the mutations identified by huber et al .   are located within the cullin homology domain ( exons 19 to 24 ) , which is responsible for roc1 binding .
biochemical characterization of the cul7 nonsense and missense mutations arg1445x ( where x indicates a stop codon ) and his1464pro , respectively , were shown to render cul7 deficient in recruiting roc1 .
arg1445x was predicted to yield a truncated cul7 polypeptide ( lacking the 254 carboxy - terminal amino acids ) , and his1464pro was predicted to introduce a structural alteration in the cullin homology domain . a study by maksimova et al .  
identified 43 patients from 37 yakut families , a geographically isolated ethnic group in russia , with a short stature syndrome similar to 3-m syndrome . a common mutation in the cul7 gene , insertion t at position 4582 in exon 25 ,
was identified that is predicted to cause a frameshift and subsequent premature stop codon at position 1553 ( q1553x ) .
given that cyclin d1   and insulin receptor substrate 1 ( irs-1 )   are potential proteolytic targets of the cul7 e3 ligase , it is tempting to speculate that either disturbed cyclin d1-dependent mechanisms or dysregulated irs-1-mediated signaling pathways might contribute to the pathomechanism of 3-m syndrome .
altogether , studies with 3-m and yakut patients , combined with proliferative defects observed in cul7 knockout mice ( table 3 ) , have strongly suggested a prominent role for cul7 in growth regulation ( reviewed by ) .
several familial mutations in the cul4b gene were associated with x - linked mental retardation syndrome ( xlmr ; omim 300639 ) .
the authors   reported three truncating , two splice - site and three missense variants at conserved amino acids in the cul4b gene on xq24 in 8 of 250 families ( 3% ) with xlmr . during adolescence of these patients a syndrome emerged with delayed puberty , hypogonadism , growth retardation , foot abnormalities , relative macrocephaly , central obesity , aggressive outbursts and seizures .
the complex phenotype of patients with cul4b mutations argues for pleiotropic roles of cul4b that remain to be determined . the cullin - based crls function through their cognate substrate - recognition molecules , such as the f - box , socs , btb and dcaf proteins ( figure 4 and table 2 ) . the f - box , socs , btb and dcaf protein families each contain a distinct motif that is recognized by an adaptor molecule , which is linked to a cognate cullin ( figure 4 ) .
for example , humans contain about 61 f - box proteins , all of which can bind to skp1 through the f - box domain . through skp1 , which binds to cul1 ( table 2 ) ,
most of the f - box proteins can thus be assembled into the scf e3 complex ( figure 4 ) .
bioinformatics studies have identified hundreds of human genes that are predicted to encode f - box , socs , btb and dcaf proteins , thereby potentially forming over 200 crls ( figure 5 ) .
although the extent of the crl family requires experimental verification , it is nonetheless reasonable to assume that crls target numerous cellular protein substrates and hence have an impact on all biological processes .
in addition , studies with f - box proteins , including -trcp , skp2 and fbw7 , demonstrate an ability of one substrate recognition protein to bind multiple substrates , thereby expanding the functional range of crls .
for recent reviews on the diverse targeting functions of these proteins , see   ( f - box family ) ,   ( btb family ) and   ( dcaf family ) .
pie chart of the numbers of human e3 ubiquitin ligases , estimated from the numerical distribution of genes predicted to encode e3 ring or hect polypeptides or , in the case of crls , the substrate - recognition molecules that include f - box , socs , btb with 3-box and dcaf proteins .
numbers refer to human gene numbers for f - box , socs , hect , u - box and non - crl ring proteins , derived from li et al . .
kay hofmann has kindly provided the tally of human genes encoding btb and 3-box proteins ( personal communication ) .
the hofmann estimation is based on the structural work from schulman and colleagues , which identified a critical role for the 3-box in the assembly of crl3 with the spop btb protein .
72 btb - only ( without 3-box ) genes have not been counted for the estimation of crl3s .
most , if not all , cullins are found covalently conjugated with an ubiquitin - like molecule , nedd8 .
this modification , termed neddylation , activates the e3 ligase activity of crls by promoting substrate polyubiquitination ( reviewed by ) .
recent studies have suggested conformation - based mechanisms that explain the activating role of neddylation . in vitro mutagenesis experiments
have suggested that the interactions between human roc1 and cul1 's carboxy - terminal tail in the unmodified state render scf inactive .
it was proposed that the conjugation of nedd8 to the residue k720 of cul1 induces drastic conformational changes in cul1 that liberate roc1 , thereby driving scf into an active state .
this hypothesis was supported by recent structural studies by schulman and colleagues   that revealed extensive conformational changes in cul5 when conjugated with nedd8 .
another activating mechanism , proposed by schulman and colleagues , suggests that the neddylation - mediated conformational changes in cullin enabled the repositioning of the ring - tethered ubiquitin - loaded e2 to a bound substrate for catalysis . in support of this model , in vitro cross - linking experiments
have revealed that neddylation brought a scf substrate into a close proximity to an e2 ubiquitin - conjugating enzyme .
neddylation is reversed by the cop9 signalosome , which enzymatically removes nedd8 from a cullin molecule .
cop9 is an eight - subunit complex that was originally identified as a suppressor of plant photomorphogenesis .
it was shown that cop9 's jab1/csn5 subunit contains a jab1/mpn domain metalloenzyme ( jamm ) motif critical for cop9 's nedd8 isopeptidase activity .
it is thus believed that crl activities are dynamically controlled by cullin neddylation - deneddylation cycles .
it was observed that an scf complex bound to a substrate contained higher levels of neddylated cul1 , suggesting that substrate - e3 interactions may trigger neddylation .
crl is also regulated by cullin - associated and neddylation - dissociated-1 ( cand1 ) , which inhibits the e3 ligase activity of crls by binding to all cullins in their un - neddylated forms .
the cul1-cand1 interaction is understood at the structural level , as the human cul1-rbx1-cand1 complex showed that cand1 binds both the cul1 amino and carboxyl termini . however
, a recent study   has revealed that only a small fraction of cullin is bound to cand1 in human cells .
by organizing crls that presumably direct numerous substrates to ubiquitin - dependent degradation , the cullin family proteins build a cellular regulatory network of fundamental importance in controlling protein homeostasis , thereby altering a wide range of biological processes , from cell cycle regulation to signal transduction .
although bioinformatics predicts a large number of crls ( figure 5 ) , we still have knowledge on only a handful of substrates . proteomics - based approaches and
newly developed global protein stability profiling technology   have proven effective in the identification of novel substrates .
however , it remains to be seen whether these methods , coupled with agents that affect cell signaling , could lead to the isolation of substrates , whose turnover rates are dictated by often transient post - translational modifications , such as phosphorylation , prolyl hydroxylation   and glycosylation . although the assembly of a majority of crls is understood in considerable detail ( table 2 and figure 4 ) , little is known about the control of the assembly in cells . it has long been suggested that the substrate - crl interactions dictate the cullin neddylation - deneddylation cycle , which turns on and off the crl 's e3 ubiquitin ligase activity .
however , the mechanism by which this is achieved is elusive . given the intricate role of cullins and crls in a multitude of biological processes , it is likely that cullin dysfunction will emerge as a pathogenetic factor in diseases .
indeed , cul7 and cul4b mutations have been identified in human disorders , but further studies are required to determine the underlying pathomechanisms .
genetic studies in organisms from yeast to mouse have revealed a prominent role for cullins and crls in cell - cycle progression ( table 3 ) .
recent advances have validated efforts in drug - targeting the ubiquitin - proteasome system , with the proteasome inhibitor bortezomib now approved for the treatment of patients with multiple myeloma or mantle cell lymphoma ( reviewed by ) . a small molecule inhibitor ( mln4924 ) suppressing
the nedd8 activating pathway is currently in clinical trials , having demonstrated success in tumor suppression in animal model studies , and two small molecule inhibitors have been identified recently to inhibit scf activities by different mechanisms .
the ubiquitin - proteasome system is a selective protein degradation pathway in which a substrate is first tagged with a chain of ubiquitin and the resulting modified protein is then recognized by the 26s proteasome , where proteolysis of the substrate takes place .
first , the chemically inert ubiquitin molecule is activated in an atp - dependent reaction by forming a thioester bond between the carboxy - terminal carboxyl group of ubiquitin and the catalytic cysteine of an e1 activating enzyme .
second , in a trans(thio)esterification reaction ubiquitin is transferred to the active site cysteine of an e2 ubiquitin - conjugating enzyme . in the last step , an e3 ubiquitin ligase functions to orchestrate the transfer of ubiquitin to a substrate protein , forming an isopeptide bond between the ubiquitin carboxy - terminal glycine residue and substrate lysine -amino group .
the action of an e3 typically involves recognition of a specific degradation motif ( degron ) on the substrate .
the human genome encodes two e1 activating enzymes , 37 e2 conjugating enzymes and more than 500 e3 ubiquitin ligases .
a substrate protein can be conjugated with just one ubiquitin ( monoubiquitination ) , one ubiquitin molecule at different lysines residues ( multiubiquitination ) or by a chain of several ubiquitin moieties ( polyubiquitination ) .
ubiquitin contains seven lysine residues ( lys6 , lys11 , lys27 , lys29 , lys33 , lys48 and lys63 ) that can act as acceptors and result in entirely different chain conformations .
for instance , although lys48-linked chains are the canonical recognition motif for the proteasome , lys63-linked chains have important non - degradative roles in cell signaling , dna - damage response and endocytosis .
monoubiquitination typically has non - proteolytic functions , such as the internalization of cell - surface receptors .
e3 ubiquitin ligases are a diverse group of enzymes that recognize both a substrate protein and an e2 ubiquitin - conjugating enzyme . e3 ubiquitin ligases can be subdivided into two major classes : hect - type and ring - type e3 ligases .
the single - molecule hect - type e3 ligases are characterized by a homologous to the e6-ap carboxyl terminus ( hect ) domain that forms a thioester intermediate with ubiquitin as a prerequisite for ubiquitin transfer to the substrate protein .
in contrast , ring - type e3 ligases use ring ( really interesting new gene)-zinc finger domains to recruit and allosterically activate an ubiquitin - charged e2 enzyme for direct ubiquitin transfer to the substrate .
ring finger domains have a characteristic architecture of three  strands , one -helical domain and two free loops that are arranged by znions .
the loops are stabilized by a cluster of cysteine residues and up to two histidines .
u - box e3 ligases are a subgroup of the ring - type e3s and contain a structurally modified ring - motif ( the u - box ) that lacks the ability to chelate znions . of about 300 ring proteins expressed in human cells , the multi - subunit cullin - ring ligase ( crl )
complexes constitute the major group and are characterized by two signature components : a cullin ( cul ) scaffold protein and the ring - finger protein roc1 or roc2 ( also known as rbx1/hrt1 or rbx2 , respectively ) .
we thank r deshaies for helping in the preparation of figure 5 and k hofmann for sharing his bioinformatics information on estimation of the number of btb proteins with a 3-box .
we thank maren hieber , kathleen meyer , wiebke schulze and benjamin wolf for critical reading of the manuscript .
as was supported by the german research foundation ( dfg ) grant sa 1706/3 - 1 and the marie curie international reintegration grant 256584 .
 summarycullin proteins are molecular scaffolds that have crucial roles in the post - translational modification of cellular proteins involving ubiquitin . 
 the mammalian cullin protein family comprises eight members ( cul1 to cul7 and parc ) , which are characterized by a cullin homology domain . 
 cul1 to cul7 assemble multi - subunit cullin - ring e3 ubiquitin ligase ( crl ) complexes , the largest family of e3 ligases with more than 200 members . 
 although cul7 and parc are present only in chordates , other members of the cullin protein family are found in drosophila melanogaster , caenorhabditis elegans , arabidopsis thaliana and yeast . 
 a cullin protein tethers both a substrate - targeting unit , often through an adaptor protein , and the ring finger component in a crl . 
 the cullin - organized crl thus positions a substrate close to the ring - bound e2 ubiquitin - conjugating enzyme , which catalyzes the transfer of ubiquitin to the substrate . 
 in addition , conjugation of cullins with the ubiquitin - like molecule nedd8 modulates activation of the corresponding crl complex , probably through conformational regulation of the interactions between cullin 's carboxy - terminal tail and crl 's ring subunit . genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective crls . 
 crls target numerous substrates and thus have an impact on a range of biological processes , including cell growth , development , signal transduction , transcriptional control , genomic integrity and tumor suppression . 
 moreover , mutations in cul7 and cul4b genes have been linked to hereditary human diseases . 
Summary
Gene organization and evolutionary history
Characteristic structural features
Localization and function
Frontiers
Box 1. The ubiquitin-proteasome system
Box 2. E3 ubiquitin ligases
Acknowledgements