The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases

doi:10.1016/S0962-8924(00)01834-1

Trends in Cell Biology

Volume 10, Issue 10, 1 October 2000, Pages 429-439

https://doi.org/10.1016/S0962-8924(00)01834-1 Get rights and content

Abstract

Recently, many new examples of E3 ubiquitin ligases or E3 enzymes have been found to regulate a host of cellular processes. These E3 enzymes direct the formation of multiubiquitin chains on specific protein substrates, and – typically – the subsequent destruction of those proteins. We discuss how the modular architecture of E3 enzymes connects one of two distinct classes of catalytic domains to a wide range of substrate-binding domains. In one catalytic class, a HECT domain transfers ubiquitin directly to substrate bound to a non-catalytic domain. Members of the other catalytic class, found in the SCF, VBC and APC complexes, use a RING finger domain to facilitate ubiquitylation. The separable substrate-recognition domains of E3 enzymes provides a flexible means of linking a conserved ubiquitylation function to potentially thousands of ubiquitylated substrates in eukaryotic cells.

Section snippets

Substrate recognition by simple and multicomponent E2–E3 complexes

After activation by an E1 enzyme, the E2 ubiquitin-conjugating enzyme and E3 ubiquitin ligase cooperate to assemble the multiubiquitin chain on a protein substrate. In studies so far, the E3 ubiquitin ligase uses protein–protein interaction domains outside the catalytic domain to bind to substrate. We discuss the architecture and substrate binding activities of the HECT domain E3 enzymes and four classes of RING finger E3 ubiquitin ligases: the SCF, VBC and anaphase-promoting (APC) complexes,

HECT domain proteins

HECT domain proteins are found in eukaryotes from yeast to humans and are defined by a 350 amino acid HECT domain (homologous to E6-AP C terminus), originally identified in E6-AP (Ref. 3). E6-AP is the cellular ubiquitin ligase recruited by the human papilloma virus E6 oncoprotein to induce degradation of the p53 tumour suppressor⁴. Within the HECT domain of E6, a conserved cysteine forms a thioester with ubiquitin. This intermediate is essential for ubiquitylation. HECT domain proteins are

SCF (Skp1, cullin, F-box) complexes

The SCF class of ubiquitin ligases contains at least four proteins: Skp1, Cul1, Roc1/Rbx1/Hrt1 and an F-box protein (see Fig. 1). SCF substrates are bound directly by adaptors called F-box proteins, which contain an ∼45-residue motif called an F-box and bind to substrates⁹ through protein–protein interaction domains (reviewed in Ref. 1). The F-box is required for binding to Skp1, a protein that is important in SCF complexes but potentially plays other roles (see below). Skp1 in turn associates

The von Hippel-Lindau (VHL)–Elongin B–Elongin C (VBC) ubiquitin ligase

The von Hippel-Lindau (VHL–Elongin B–Elongin C (or VBC) ubiquitin ligase contains the VHL tumour suppressor, which is inactivated in von Hippel-Lindau disease and in over 80% of sporadic renal cell carcinomas1, 36. The VBC ubiquitin ligase is structurally similar to the SCF E3 complex (Fig. 1). It contains Roc1/Rbx1, the cullin Cul2, a protein homologous to Skp1 called elongin C, a probable adaptor protein called VHL and another factor called elongin B (Fig. 1). VHL binds to elongin C through a

The anaphase-promoting complex (APC) ubiquitin ligase

The APC was the first multicomponent ubiquitin ligase described and is required for the degradation of substrates controlling the metaphase-to-anaphase transition and the destruction of cyclin B to allow the exit from mitosis42, 43, 44. Similar to the SCF and VBC complexes, the APC contains a cullin homologue, Apc2, and a RING-H2 finger protein similar to Roc1/Rbx1, called Apc11. Also, like the SCF, the APC associates with proteins that activate the APC towards specific substrates⁴². Two

Single protein RING finger (SPRF) E3 ubiquitin ligases

The identification of RING finger proteins in SCF complexes and other E3 proteins suggests the broad use of these domains for ubiquitylation⁵⁶. As stated earlier, RING fingers are zinc-binding domains with a defined octet of cysteine and histidine residues. The subtype RING-H2 domain contains histidines at positions 4 and 5 (see Fig. 2) and is the form found in Roc1/Rbx1, Roc2, Apc11 and other ubiquitin ligases. Another subtype RING-HC (or C3HC4) contains only one histidine (at position 4) and

Catalysis

Little is known about the catalytic mechanisms employed by the various E3 ubiquitin ligases. Apart from the HECT domain ubiquitin ligases, which accept ubiquitin from E2 enzymes to create a thioester bond and directly catalyse ubiquitin transfer to the target protein62, 63, no other ubiquitin ligases are currently thought to form thioester bonds with ubiquitin. Because the common feature of non-HECT domain ubiquitin ligases is the RING finger, this domain appears central to catalysis. The

Regulation of activity of E3 complexes by ubiquitin-like molecules

Some examples of how modifications or other factors regulate ubiquitin ligase complexes are known. SCF ubiquitin ligases have been found to be regulated by phosphorylation at least at the level of substrate recognition or subunit recruitment (as discussed above), through posttranslational modification by other ubiquitin-like molecules (UBLs) and by accessory factors, including targeting to subcellular localizations and assembly into multiprotein complexes (for reviews, see 69, 70). Because of

Summary: different mechanisms for ubiquitin ligase activity?

The recent discoveries defining the functional domains of known ubiquitin ligases suggest a simple model where the RING or HECT catalytic domains are tethered to specific substrates by protein–protein recognition domains. This tether appears to facilitate formation of the multiubiquitin chain, although the mechanisms remain unclear.

Why some ubiquitin ligases use HECT domains, which form a thioester-linked ubiquityl intermediate, and others RING fingers, which apparently do not, also remains

Uncited reference

Acknowledgements

We apologize to many authors who we could not cite because of space constraints. This work was supported in part by grants from the NIH [GM54811 and GM60439], Medical Scientist Training Program (to J.D.R.R.) and Cancer Biology Training grants (to L.F. and B.K.K.) and from the Howard Hughes Medical Institute (to P.K.J., A.G.E., and J.Y.H.).

References (76)

R.J. Deshaies
SCF and Cullin/Ring H2-based ubiquitin ligases
Annu. Rev. Cell Dev. Biol.
(1999)
M. Scheffner
Ubiquitin, E6-AP, and their role in p53 inactivation
Pharmacol. Ther.
(1998)
M. Scheffner
The HPV-16 E6 and E6-AP complex functions as a ubiquitin-protein ligase in the ubiquitination of p53
Cell
(1993)
G. Wang
Functional domains of the Rsp5 ubiquitin-protein ligase
Mol. Cell. Biol.
(1999)
J.D. Singer
Cullin-3 targets cyclin E for ubiquitination and controls S phase in mammalian cells
Genes Dev.
(1999)
J.H. Seol
Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34
Genes Dev.
(1999)
D. Skowyra
F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex
Cell
(1997)
C. Cenciarelli
Identification of a family of human F-box proteins
Curr. Biol.
(1999)
J.T. Winston
A family of mammalian F-box proteins
Curr. Biol.
(1999)
D.M. Stone
Characterization of the human suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli
J. Cell Sci.
(1999)

K. Kominami

Two F-box/WD-repeat proteins Pop1 and Pop2 form hetero- and homo-complexes together with cullin-1 in the fission yeast SCF (Skp1-Cullin-1-F-box) ubiquitin ligase

Genes To Cells

(1998)

I.D. Russell

The unstable F-box protein p58-Ctf13 forms the structural core of the CBF3 kinetochore complex

J. Cell Biol.

(1999)

W.G. Kaelin et al.

The VHL tumour-suppressor gene paradigm

Trends Genet.

(1998)

J.G. Zhang

The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation

Proc. Natl. Acad. Sci. U. S. A.

(1999)

D.A. Wolf et al.

Cell cycle: oiling the gears of anaphase

Curr. Biol.

(1998)

W. Zachariae

Mass spectrometric analysis of the anaphase-promoting complex from yeast: identification of a subunit related to cullins

Science

(1998)

M. Glotzer

Cyclin is degraded by the ubiquitin pathway

Nature

(1991)

S.L. Jaspersen

Inhibitory phosphorylation of the APC regulator Hct1 is controlled by the kinase Cdc28 and the phosphatase Cdc14

Curr. Biol.

(1999)

W. Zachariae

Control of cyclin ubiquitination by CDK-regulated binding of Hct1 to the anaphase promoting complex

Science

(1998)

C.A. Joazeiro

The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase

Science

(1999)

S. Fang

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

J. Biol. Chem.

(2000)

R. Honda et al.

Activity of MDM2, a ubiquitin ligase, toward p53 or itself is dependent on the RING finger domain of the ligase

Oncogene

(2000)

J.M. Huibregtse

A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase

Proc. Natl. Acad. Sci. U. S. A.

(1995)

K.L. Lorick

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination

Proc. Natl. Acad. Sci. U. S. A.

(1999)

C. Ptak

Functional and physical characterization of the cell cycle ubiquitin-conjugating enzyme CDC34 (UBC3). Identification of a functional determinant within the tail that facilitates CDC34 self-association

J. Biol. Chem.

(1994)

M. Tyers et al.

Proteolysis and the cell cycle: with this RING I do thee destroy

Curr. Opin. Genet. Dev.

(2000)

K.L. Craig et al.

The F-box: a new motif for ubiquitin dependent proteolysis in cell cycle regulation and signal transduction

Prog. Biophys. Mol. Biol.

(1999)

E.T. Yeh

Ubiquitin-like proteins: new wines in new bottles

Gene

(2000)

J.M. Desterro

SUMO-1 modification of IκBα inhibits NF-κB activation

Mol. Cell

(1998)

T. Buschmann

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53

Cell

(2000)

R.M. Feldman

A complex of Cdc4p, Skp1p and Cdc53p/cullin catalyzes ubiquitination of the phophorylated CDK inhibitor Sic1p

Cell

(1997)

R.J. Deshaies

Ubiquitination of the G1 cyclin Cln2p by a Cdc34p-dependent pathway

EMBO J.

(1995)

M. Tyers et al.

Proteolysis and the cell cycle: with this RING I do thee destroy

Curr. Opin. Genet. Dev.

(2000)

M. Sudol

From Src Homology domains to other signaling modules: proposal of the ‘protein recognition code’

Oncogene

(1998)

D. Rotin

WW (WWP) domains: from structure to function

Curr. Top. Microbiol. Immunol.

(1998)

H. Zhu

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation

Nature

(1999)

C. Bai

SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box

Cell

(1996)

T. Kamura

Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase

Science

(1999)

Cited by (563)

Proteostasis in aging-associated ocular disease
2022, Molecular Aspects of Medicine
Vision impairment has devastating consequences for the quality of human life. The cells and tissues associated with the visual process must function throughout one's life span and maintain homeostasis despite exposure to a variety of insults. Maintenance of the proteome is termed proteostasis, and is vital for normal cellular functions, especially at an advanced age. Here we describe basic aspects of proteostasis, from protein synthesis and folding to degradation, and discuss the current status of the field with a particular focus on major age-related eye diseases: age-related macular degeneration, cataract, and glaucoma. Our intent is to allow vision scientists to determine where and how to harness the proteostatic machinery for extending functional homeostasis in the aging retina, lens, and trabecular meshwork. Several common themes have emerged despite these tissues having vastly different metabolisms. Continued exposure to insults, including chronic stress with advancing age, increases proteostatic burden and reduces the fidelity of the degradation machineries including the ubiquitin-proteasome and the autophagy-lysosome systems that recognize and remove damaged proteins. This “double jeopardy” results in an exponential accumulation of cytotoxic proteins with advancing age. We conclude with a discussion of the challenges in maintaining an appropriate balance of protein synthesis and degradation pathways, and suggest that harnessing proteostatic capacities should provide new opportunities to design interventions for attenuating age-related eye diseases before they limit sight.
Characterization of TRIM16, a member of the fish-specific finTRIM family, in olive flounder Paralichthys olivaceus
2022, Fish and Shellfish Immunology
Citation Excerpt :
PoTRIM16 mRNA levels fluctuated markedly during early development. In addition, fish embryos and hatchlings are exposed to pathogens long before they are able to develop a mature immune response and while no immunoglobulins are present [24–26]. Therefore, our results provide insight into the development of the innate immune response in fish eggs.
Tripartite motif-containing (TRIM) proteins are conserved throughout the metazoan kingdom, and the TRIM subset finTRIM is highly diversified in fish. We isolated TRIM16 cDNA, a member of the finTRIM family, from the olive flounder Paralichthys olivaceus (PoTRIM16). PoTRIM16 contained a 1,725-bp coding sequence encoding a 574-amino acid polypeptide, which in turn contained a really interesting new gene (RING) finger domain, B-box-type zinc finger (B-BOX), nuclease SbcCD subunit C (SbcC), structural maintenance of chromosome (SMC prok B), and stonustoxin (SNTX) subunit alpha (SPRY-PRY-SNTX). Multiple alignment of related sequences revealed that PoTRIM16 showed 86.63–97.40% identity with fish orthologues, and a phylogenetic tree was constructed of vertebrates. PoTRIM16 mRNA was detected in all tissues examined; levels were highest in the eye and ovary. PoTRIM16 mRNA expression was investigated during early development. Under VHSV infection, PoTRIM16 mRNA was downregulated in the liver of P. olivaceus. This is the first study to characterize fish-specific finTRIM in P. olivaceus, which may play a role in the immune response against virus infection.
E3 ubiquitin ligase RNF114 promotes vesicular stomatitis virus replication via inhibiting type I interferon production
2022, Microbial Pathogenesis
As a single-stranded RNA virus, vesicular stomatitis virus (VSV) causes influenza-like clinical symptoms in infected individuals. Type-I interferon signaling pathway plays a vital role in inhibiting VSV replication. It has been shown that RNF114 (RING finger protein 114) acts as an E3 ubiquitin ligase to regulate the type-I interferon signaling pathway. In contrast, the effects of RNF114 from Chinese sturgeon or sea perch remain controversial. In the present study, we reported the effect of human RNF114 on VSV infection. Overexpression of RNF114 promoted VSV replication, while depletion of RNF114 reduced viral replication. We further found that RNF114 inhibited type-I interferon production via interacting with mitochondrial antiviral signaling protein (MAVS). Moreover, in vivo experiments demonstrated that RNF114 could also accelerate VSV replication and virus-induced inflammation in lung tissues. Collectively, our findings supported that RNF114 negatively regulated the type-I interferon signaling pathway during VSV replication, providing novel and favorable insights into clinical treatment of VSV infection.
Post-translational modifications inducing proteasomal degradation to counter HIV-1 infection
2020, Virus Research
Post-translational modifications (PTMs) are integral to regulating a wide variety of cellular processes in eukaryotic cells, such as regulation of protein stability, alteration of celluar location, protein activity modulation, and regulation of protein interactions. HIV-1, like other eukaryotic viruses, and its infected host exploit the proteasomal degradation system for their respective proliferation and survival, using various PTMs, including but not limited to ubiquitination, SUMOylation, NEDDylation, interferon-stimulated gene (ISG)ylation. Essentially all viral proteins within the virions -- and in the HIV-1-infected cells -- interact with their cellular counterparts for this degradation, utilizing ubiquitin (Ub), and the Ub-like (Ubl) modifiers less frequently, to eliminate the involved proteins throughout the virus life cycle, from the entry step to release of the assembled virus particles. Such interplay is pivotal for, on the one hand, the cell to restrict proliferation of the infecting virus, and on the other, for molecular counteraction by the virus to overcome this cellular protein-imposed restriction. Recent reports indicate that not only viral/cellular proteins but also viral/viral protein interactions play vital roles in regulating viral protein stability. We hence give an overview of the molecular processes of PTMs involved in proteasomal degradation of the viral and cellular proteins, and the viral/viral and viral/cellular protein interplay in restriction and competition for HIV-1 vs. host cell survival. Insights in this realm could open new avenues for developing therapeutics against HIV-1 via targeting specific steps of the proteasome degradation pathway during the HIV-1 life cycle.
The effect of TRIM5 variants on the susceptibility to HIV-1 infection and disease progression in the Polish population
2024, Annals of Human Genetics
The Auto-Regulation of ATL2 E3 Ubiquitin Ligase Plays an Important Role in the Immune Response against Alternaria brassicicola in Arabidopsis thaliana
2024, International Journal of Molecular Sciences

View all citing articles on Scopus

View full text

Trends in Cell Biology

ReviewThe lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases

Abstract

Section snippets

Substrate recognition by simple and multicomponent E2–E3 complexes

HECT domain proteins

SCF (Skp1, cullin, F-box) complexes

The von Hippel-Lindau (VHL)–Elongin B–Elongin C (VBC) ubiquitin ligase

The anaphase-promoting complex (APC) ubiquitin ligase

Single protein RING finger (SPRF) E3 ubiquitin ligases

Catalysis

Regulation of activity of E3 complexes by ubiquitin-like molecules

Summary: different mechanisms for ubiquitin ligase activity?

Uncited reference

Acknowledgements

Annu. Rev. Cell Dev. Biol.

Pharmacol. Ther.

Cell

Mol. Cell. Biol.

Genes Dev.

Genes Dev.

Cell

Curr. Biol.

Curr. Biol.

J. Cell Sci.

Genes To Cells

J. Cell Biol.

Trends Genet.

Proc. Natl. Acad. Sci. U. S. A.

Curr. Biol.

Science

Nature

Curr. Biol.

Science

Science

J. Biol. Chem.

Oncogene

Proc. Natl. Acad. Sci. U. S. A.

Proc. Natl. Acad. Sci. U. S. A.

J. Biol. Chem.

Curr. Opin. Genet. Dev.

Prog. Biophys. Mol. Biol.

Gene

Mol. Cell

Cell

Cell

EMBO J.

Proteolysis and the cell cycle: with this RING I do thee destroy

Curr. Opin. Genet. Dev.

From Src Homology domains to other signaling modules: proposal of the ‘protein recognition code’

Oncogene

WW (WWP) domains: from structure to function

Curr. Top. Microbiol. Immunol.

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation

Nature

SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box

Cell

Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase

Science

Review
The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases