Lennarz W.J., Lane M.D. (eds.) Encyclopedia of Biological Chemistry. Four-Volume Set . V. 4

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that interacted with baits contingent upon the modiﬁ-

cation of the bait by a coexpressed kinase. A second

point of interest was the feasibility of identifying

proteins that interacted not with a single protein, but

with a complex formed by a bait and one or more

simultaneously expressed “co-baits.” A number of

investigators have used a two-hybrid approach to

identify proteins that interact as constituents of ternary

or even quarternary complexes.

Another application of interest has been to use the

two-hybrid system to identify and characterize inter-

actions between proteins and non-protein ligands.

To date, two-hybrid derivative systems have been used

to score interactions between proteins and small

molecules/drugs, and proteins and RNAs. In each case,

the derivative system has been shown to function at the

level of library screening. While screening for RNAs has

become relatively broadly adapted as a research tool,

screening for drugs that regulate protein–protein

interactions remains at an earlier stage of validation.

Detailed description of the derivatization of the two-

hybrid system required for the screening of non-protein

ligands is beyond the scope of this review.

The two-hybrid system has been used by a number of

investigators to identify peptide “aptamers” capable of

binding and regulating the functions of speciﬁc bait

proteins. Peptides isolated based on two-hybrid selection

have been found capable of regulating diverse functions

of their cognate baits, and may provide the basis for

design of peptidomimetics or small molecule derivatives

of therapeutic value. This is currently an area of

active investigation.

Finally, a recent effort has been to reﬁne the two-

hybrid system so as to make it a more useful tool for

evaluating the selectivity of protein interactions. In these

approaches, the two-hybrid system is “parallelized,”

using two discrete sets of baits and reporters within a

single strain of yeast. In one example, bait 1 (a fusion to

LexA) directs the expression of the lacZ and LEU2

reporters, while bait 2 (a fusion to cI) directs the

expression of gusA (colorimetric) and LYS2 (auxo-

trophic) reporters. In a situation where a prey initially

interacts with both baits 1 and 2, turning on all four

reporters, mutagenesis of the prey can be coupled with

selective screening for activation of the lacZ and LEU2,

versus the gusA and LYS2, reporters, providing a

convenient means of identifying altered speciﬁcity

mutants that can be used to deconvolute signal

transduction pathways.

Alternative Two-Hybrid Systems

Although the yeast two-hybrid system has found many

useful applications, it has not been appropriate in all

cases. Toward the goal of identifying small molecules or

mutations that disrupt, rather than permit, protein –

protein interactions, a “reverse” two-hybrid system has

been developed. In this system, activation of a reporter

gene is toxic, providing selection pressure for clones that

have lost protein–protein interactions. For proteins that

strongly activate transcription in yeast, and hence

cannot be used to create baits, one solution has been

to move the two-hybrid components to an RNA

polymerase III-based reporter system, eliminating tran-

scriptional activity. Other investigators have generated

two-hybrid-like systems in bacteria, where eukaryotic

transcriptional activation sequences are generally

ineffective. For proteins that are membrane associated,

or cannot efﬁciently be nuclear localized, several

methods for assessing protein interaction at membranes

or in the cytoplasm have been developed. These include

systems in which interaction of the bait and prey

reconstitutes an intracellular signaling pathway (e.g.,

the SOS system), or generate an assayable enzymatic

by-product, as in the ubiquitin-based split-protein

sensor system (USPS). In one recent and promising

development, the USPS system has been further modiﬁed

such that interactions of a bait and prey at the cell

membrane triggers the cleavage and release of a

transcription factor that migrates to the nucleus and

activates standard yeast two-hybrid colorimetric and

auxotrophic reporters. This approach combines advan-

tages of membrane-based interaction detection with the

ability to use well-validated screening modalities.

Role in Proteomics

Two hybrid protein –protein interaction systems are an

important element of strategies to analyze the complete

protein–protein interactions of proteomes. To date,

extensive protein interaction maps have been con-

structed for viruses and yeast, and large-scale screening

efforts are underway in Caenorhabditis elegans

(C. elegans), Drosophila, and humans. These efforts

provide a useful complement to simultaneous efforts to

analyze protein interactions by mass spectrometry:

whereas mass spectrometry is of value in identifying

proteins which bind with high afﬁnity to an assembled

complex, albeit potentially weakly to any single inter-

active partner, the yeast two-hybrid system excels at

identifying proteins that interact with high afﬁnity with

single deﬁned partners. Interactions being detected by

these means are being integrated into datasets includ-

ing transcriptional expression proﬁles, genetic inter-

action data, and protein localization data for the same

proteins, providing a fundamental tool for the emerging

ﬁeld of systems biology. It is likely that continuing

application of the two-hybrid protein– protein inter-

action will remain a source of scientiﬁc insights for many

years to come.

292

TWO-HYBRID PROTEIN–PROTEIN INTERACTIONS

FURTHER READING

Bernstein, D. S., Buter, N., Stumpf, C., and Wickens, M. (2002).

Analyzing mRNA–protein complexes using a yeast three-hybrid

system. Methods 26, 123– 141.

Fashena, S. J., Serebriiskii, I. G., and Golemis, E. A. (2000).

The continued evolution of hybrid screening approaches in

yeast: How to outwit different baits with different preys. Gene

250, 1–14.

Fields, S., and Song, O. (1989). A novel genetic system to detect

protein–protein interaction. Nature 340, 245–246.

Schwikowski, B., Ideker, T., and Uetz, P. (2002). Visualization and

integration of protein– protein interactions. In Protein Interactions

(E. A. Golemis, ed.) Cold Spring Harbor Laboratory Press, Cold

Spring Harbor, NY.

Serebriiskii, I. G., Khazak, V., and Golemis, E. A. (2001). Redeﬁnition

of the yeast two-hybrid system in dialogue with changing priorities

in biological research. BioTechniques 30, 634–655.

Stagljar, I., and Fields, S. (2002). Analysis of membrane protein

interactions using yeast-based technologies. Trends Biochem. Sci.

27, 559–563.

Walhout, A. J., Reboul, J., Shtanko, O., Bertin, N., Vaglio, P., Ge, H.,

Lee, H., Doucette-Stamm, L., Gunsalus, K. C., Schetter, A. J.,

Morton, D. G., Kemphues, K. J., Reinke, V., Kim, S. K., Piano, F.,

and Vidal, M. (2002). Integrating interactome, phenome, and

transcriptome mapping data for the C. elegans Germline. Curr.

Biol. 12, 1952–1958.

Zhu, H., and Snyder, M. (2002). “Omic” approaches for unraveling

signaling networks. Curr. Opin. Cell Biol. 14, 173– 179.

BIOGRAPHY

Ilya Serebriiskii is a Staff Scientist in the Division of Basic Sciences at

the Fox Chase Cancer Center in Philadelphia, PA. His research

interests include bacterial and yeast genetics and proteomics. He has

created and evaluated numerous advanced protein interaction screen-

ing tools based on two-hybrid paradigms. He holds a Ph.D. in Biology

from VNIIGenetika, Moscow, Russia. He received his postdoctoral

training with Prof. Gerard Leblon at The Institute of Genetics

and Microbiology, University of Paris-Sud, France, and then with

Dr. Erica Golemis at Fox Chase Cancer Center.

Erica Golemis is a Member of the Division of Basic Sciences at Fox

Chase Cancer Center. Her interests include the study of protein

interactions, and the intersection of cell attachment and cell cycle

control signaling in mammalian cancer development. She performed

research with Dr. Nancy Hopkins leading to a Ph. D. in Biology from

the Massachusetts Institute of Technology in Cambridge, MA.

Subsequent postdoctoral work with Dr. Roger Brent at Massachusetts

General Hospital in Boston, MA, led to the development of an original

LexA-based two-hybrid system. She has worked extensively in the area

of yeast two-hybrid system reagent development.

TWO-HYBRID PROTEIN–PROTEIN INTERACTIONS 293

Tyrosine Sulfation

Denis Corbeil and Wieland B. Huttner

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

The O-sulfation of tyrosine residues of membrane and

secretory proteins that transit through the secretory pathway

of eukaryotic cells is a ubiquitous posttranslational modiﬁ-

cation conserved in all multicellular organisms. Tyrosine

sulfation is catalyzed by tyrosylprotein sulfotransferase

(TPST) isoenzymes, which are integral membrane proteins of

the trans-Golgi network. Tyrosine sulfation has been shown to

be important for protein–protein interactions occurring in

diverse biological processes, ranging from the receptor binding

of regulatory peptides to the interaction of viral envelope

proteins with the cell surface.

Tyrosine-Sulfated Proteins

OCCURRENCE

Sulfation is one of the most abundant posttranslational

modiﬁcations of tyrosine, since up to 1% of the tyrosine

residues of total protein in an organism can be sulfated.

Tyrosine is the only amino acid residue in proteins known

to undergo sulfation. Following the ﬁrst description of a

sulfated-tyrosine residue in a peptide derived from

ﬁbrinogen by Bettelheim in 1954, it has been known

since 1982 that tyrosine-sulfated proteins occur in all

animals, from lower invertebrates up to humans.

Tyrosine-sulfated proteins also exist in the plant king-

dom, for example, in the green alga volvox, one of the

earliest truly multicellular organisms, or in higher plants

such as rice. While occurring throughout metazoan

evolution, tyrosine-sulfated proteins appear to be absent

in unicellular eukaryotes and prokaryotes, implicating

this post translational modiﬁcation in some aspect of

multicellularity.

In a given animal, tyrosine-sulfated proteins have been

observed in all tissues examined. Each tissue appears to

contain a characteristic set of tyrosine-sulfated proteins,

suggesting that proteins with tissue-speciﬁc expression

are major targets for tyrosine sulfation. In cell culture,

tyrosine sulfation of proteins has been detected in all

primary cultures and cell lines investigated, including

various secretory cells, epithelial cells, ﬁbroblasts,

neuronal cells, and cells of the immune system.

Tyrosine-sulfated proteins can be identiﬁed by var-

ious methods, including labeling using radioactive

sulfate followed by tyrosine sulfate analysis of a given

protein. In line with the intracellular localization of

tyrosylprotein sulfotransferase (TPST) in the trans-Golgi

network, all known tyrosine-sulfated proteins are either

secretory or plasma membrane proteins. Reviews with

comprehensive lists of tyrosine-sulfated proteins have

been published, and several of these proteins have been

shown to play important biological roles.

STRUCTURAL DETERMINANTS

TYROSINE SULFATION

The recognition, by TPST, of the tyrosine residue to be

sulfated in a secretory protein or the extracellular domain

of a membrane protein requires the presence of certain

structural features. These have been deduced from the

comparison of identiﬁed tyrosine sulfation sites and the

in vitro tyrosine sulfation of synthetic peptides. Although

no strict consensus sequence for tyrosine sulfation exists,

all sequences exhibit the presence of acidic amino acid

residues in the vicinity, i.e., positions 2 5 (N-terminal) to

þ5 (C-terminal), of the sulfated tyrosine residue. A

particularly critical position appears to be amino-

terminal (2 1) to the tyrosine. Turn-inducing amino

acids (P, G) are also frequently present. The few examples

of tyrosine sulfation sites lacking proline and glycine are

located near the N- or C-terminus of the protein and/or

contain several of the three other amino acid residues (D,

S, N) with signiﬁcant turn-conformational potential.

Finally, another characteristic feature common to most

identiﬁed tyrosine sulfation sites is the absence of cysteine

residues or potential N-glycosylation sites (NXS or NXT)

in the vicinity (positions 2 7toþ7). In either case, the

presence of a disulﬁde-bridge or N-linked oligosacchar-

ides is likely to prevent sulfation of nearby tyrosine

residues due to steric hindrance.

Tyrosylprotein Sulfotransferase

(EC 2.8.2.20)

THE TYROSINE SULFATION REACTION

The sulfate transfer reaction to tyrosine residues is

catalyzed by TPST, ﬁrst described in 1983, and used as a

sulfate donor, the cosubstrate 3

-phosphoadenosine

-phosphosulfate (PAPS) (Figure 1B). The transfer is

thought to occur in an ordered reaction mechanism that

involves the following sequential steps: (1) cosubstrate

PAPS binding; (2) substrate binding; (3) sulfate transfer;

(4) tyrosine-sulfated product release and, (5) PAP

release. TPST activity can be detected by incubation of

appropriate membrane preparations with [

S]PAPS,

using either endogenous or exogenous protein substrate.

Biologically, tyrosine sulfation appears to be an irre-

versible event in vivo due to the lack of a sulfatase

capable of catalyzing the desulfation of tyrosine-sulfated

proteins under physiological conditions.

PROPERTIES OF TPST

TPST is an integral membrane protein residing in the

trans-Golgi network (Figure 1A). The TPST protein can

FIGURE 1 (A) Tyrosine sulfation occurs in the trans-Golgi network (TGN). (B) TPST, a type II transmembrane protein, catalyzes the transfer of

the sulfate group (boxes) from the co-substrate PAPS to tyrosine residues of secretory proteins and ectodomains of membrane proteins passing

through the lumen of the trans-Golgi network. (C) The three principal types of tyrosine sulfation-dependent protein–protein interactions.

TYROSINE SULFATION 295

be identiﬁed in membrane preparations by MSC

(modiﬁcation after substrate cross-linking) labeling,

which is based on the cross-linking of a substrate

peptide to TPST followed by intramolecular [

S]sulfate

transfer from the cosubstrate PAPS. Various nonionic

detergentscanbeusedtosolubilizeTPSTfrom

membranes.

TPST puriﬁed from bovine adrenal medulla is a

50–54 kDa sialoglycoprotein with an apparent S-value

of 6. Its pH optimum is between 6.0 and 6.5, which is in

line with the slightly acidic pH of the trans-Golgi

network. The catalytic activity of the enzyme, towards

endogenously as well as exogenously added proteins, is

stimulated by divalent cations (Mg

2þ

,Mn

2þ

), and

ﬂuoride ions are required for maximal activity. The K

for the cosubstrate PAPS is 1.4 mM. Like most sulfo-

transferases, TPST is inhibited by PAP. In vitro, the

activity of the enzyme is also inhibited by certain lipids

such as sphingosine, but the physiological relevance of

this observation is unknown. Synthetic inhibitors of

TPST (with IC

values of 30 –40 mM) have been

generated by a combinatorial target-guided ligand

assembly technique.

The K

for most peptides (8–13 amino acid residues)

with a single tyrosine sulfation site is in the range of

10–100 mM. It is likely that similar values hold true for

individual tyrosine sulfation sites in proteins. Interest-

ingly, K

values for synthetic substrates containing

multiple tyrosine sulfation sites are considerably lower.

Given that several proteins exist with multiple adjacent

sulfation sites, e.g., cionin, heparin cofactor, and prepro-

cholecystokinin, the increased afﬁnity of TPST for such

substrates might be of physiological signiﬁcance, e.g., by

promoting stoichiometric sulfation. A remarkable

example of the sequential sulfation of four tyrosine

residues within a short stretch of amino acids is the

N-terminal domain of the CC-chemokine receptor 5

(CCR5), which is a coreceptor for HIV.

MOLECULAR CLONING AND MEMBRANE

TOPOLOGY OF TPST

Distinct TPST isoenzymes, anticipated from the differ-

ential substrate speciﬁcities of various TPST prep-

arations, exist in the human and mouse genome. Each

contains two TPSTs, TPST-1 (see Swiss-Prot Database,

Accession No. 060507) and TPST-2 (see Swiss-Prot

Database, Accession No. 060704), which were ﬁrst

characterized at the molecular level in 1998. The human

TPST-1 (7q11) and TPST-2 (22q12.1) genes encode for

370- and 377-amino acid proteins, respectively, that

share an overall 65% identity. The murine tpst genes are

located on chromosome 5, and the corresponding

proteins show , 95% identity to their human counter-

part. Both TPST isoenzymes are predicted to have a type

II membrane topology, with a very short NH

-terminal

cytoplasmic domain (8 residues) and the bulk of the

polypeptide, which is responsible for the catalytic

activity, being located in the Golgi lumen (see Figures

1A and 1B). Although overall, TPST isoenzymes display

a low degree of amino acid identity to other cytosolic

and Golgi-associated sulfotransferases, most of the

residues involved in PAPS binding, as deduced from

comparison with the crystal structure of estrogen

sulfotransferase, are conserved. The luminal domain of

either isoenzyme also contains two potential N-glyco-

sylation sites, consistent with presence of N-linked

glycans in the TPST protein.

In agreement with the occurrence of tyrosine sulfa-

tion in all metazoan species, cDNAs that predict

proteins obviously related to mammalian TPSTs are

found in various vertebrates and invertebrates, including

ﬁsh (GenBank Accession No. BI846282), frog (GenBank

Accession No. BG815875), chicken (GenBank Acces-

sion Nos. BU142429, BU217098), ﬂy (GenBank Acces-

sion No. AY124548), and worm.

TPST-1 VERSUS TPST-2

TPST-1 and TPST-2 transcripts are found in all tissues

examined, e.g., brain, heart, skeletal muscle, gut, kidney,

liver, lung, and leukocytes. Although ubiquitously

expressed, the tissue distribution of TPST-1 and TPST-

2 are not identical, recombinant TPST-1 and TPST-2

show similar, but not identical, activities toward certain

small peptide substrates, consistent with some func-

tional redundancy as well as a certain degree of

differential substrate speciﬁcity.

TPST-1- AND TPST-2-DEFICIENT MICE

TPST-1- and TPST-2-deﬁcient mice, generated by

targeted disruption of the tpst-1 and tpst-2 genes, also

point to functional redundancy between the two

isoenzymes, as either line of knock-out mice is viable

(K.L. Moore, personal communication). Although

TPST-1

2/2

animals appear normal, their body weight

is reduced about 5% and an increase in postimplanta-

tion fetal death is observed, suggesting that unidentiﬁed

proteins involved in regulation of body weight and

reproductive physiology require tyrosine sulfation for

optimal function. TPST-2 deﬁcient mice show a transi-

ent delay in growth during postnatal development. In

addition, the males, but not females, appear to be

infertile. Together, these observations are consistent with

the idea that TPST-1 and TPST-2 have distinct, but

partially overlapping, physiological roles.

296

TYROSINE SULFATION

Physiological Function

and Medical Relevance

For most tyrosine-sulfated proteins, the physiological

function of this posttranslational modiﬁcation is pres-

ently unknown. With regard to the cases in which the

biological role of tyrosine sulfation of a particular

protein has been elucidated, the common denominator

has emerged that tyrosine sulfation promotes extra-

cellular protein–protein interactions. In line with the

identiﬁcation of numerous secretory and plasma mem-

brane proteins that are tyrosine-sulfated, paradigmatic

examples exist showing that tyrosine sulfation promotes

the interaction between (1) a secretory and a plasma

membrane protein, (2) two secretory proteins, or (3) two

plasma membrane proteins (Figure 1C).

The regulatory peptide cholecystokinin is a classical

example where tyrosine sulfation of a secretory protein

dramatically promotes its interaction with a plasma

membrane protein, i.e., its cell surface receptor. Thus,

sulfated cholecystokinin is 260 times more potent than

its unsulfated form. Of the several examples of tyrosine

sulfation promoting the interaction between two

secretory proteins, the case of the binding of the

tyrosine-sulfated blood coagulation factor VIII to von-

Willebrand-factor is particularly intriguing, as it also

documents the medical relevance of this posttransla-

tional modiﬁcation. Humans with a mutation in the

critical tyrosine residue of factor VIII that is sulfated and

involved in its binding to von-Willebrand-factor are

afﬂicted with hemophilia A.

An example of tyrosine sulfation promoting the

interaction between two plasma membrane proteins is

the important role of this posttranslational modiﬁcation

for the high-afﬁnity binding of leukocyte-associated P-

selectin glycoprotein ligand (PSGL)-1 to P-selectin on

activated endothelial cells. This crucial interaction

initiates adhesion of leukocytes to the vascular wall

during inﬂammation. Tyrosine sulfation also occurs in

seven-transmembrane-segment chemokine receptors,

e.g., CCR5. Under physiological conditions, these

plasma membrane proteins play a central role in

chemokine signalling pathway through G proteins.

Remarkably, human and simian immunodeﬁciency

viruses use CCR5 as a co-receptor, together with CD4,

to mediate their attachment to the host cell membrane.

Speciﬁcally, sulfation of tyrosine residues in the CCR5

N-terminal domain has been shown to be critical for the

interaction of this protein with HIV envelope glyco-

protein gp120, leading to HIV infection. Thus, the

design of tyrosine-sulfated peptide competitors –

mimicking HIV gp120-binding sites – could turn out

to be the basis for new therapeutic compounds that will

block HIV cellular entry. These examples highlight the

medical relevance of protein tyrosine sulfation.

FURTHER READING

Bettelheim, F. R. (1954). Tyrosine-O-sulfate in a peptide from

ﬁbrinogen. J. Am. Chem. Soc. 76, 2838–2839.

Huttner, W. B. (1982). Sulphation of tyrosine residues – a widespread

modiﬁcation of proteins. Nature (London) 299, 273– 276.

Huttner, W. B. (1984). Determination and occurrence of tyrosine

O-sulfate in proteins. Meth. Enzymol. 107, 200–223.

Huttner, W. B., and Baeuerle, P. A. (1988). Protein sulfation on

tyrosine. Mod. Cell Biol. 6, 97– 140.

Huttner, W. B., Niehrs, C., and Vannier, C. (1991). Bind or bleed. Curr.

Biol. 1, 309 –310.

Kehoe, J. W., and Bertozzi, C. R. (2000). Tyrosine sulfation: A

modulator of extracellular protein–protein interactions. Chem.

Biol. 7, R57 –R61.

Kehoe, J. W., Maly, D. J., Verdugo, D. E., Armstrong, J. I., Cook, B. N.,

Ouyang, Y. B., Moore, K. L., Ellman, J. A., and Bertozzi, C. R.

(2002). Tyrosylprotein sulfotransferase inhibitors generated by

combinatorial target-guided ligand assembly. Bioorg. Med. Chem.

Lett. 12, 329–332.

Moore, K. L. (2003). The biology and enzymology of protein tyrosine

O-sulfation. J. Biol. Chem. 278, 24243–24246.

Niehrs, C., Beisswanger, R., and Huttner, W. B. (1994). Protein tyrosine

sulfation, 1993 – an update. Chem. Biol. Interact. 92, 257–271.

Ouyang, Y.-B., Crawley, J. T. B., Aston, C. E., and Moore, K. L.

(2002). Reduced body weight and increased postimplantation fetal

death in tyrosylprotein sulfotransferase-1-deﬁcient mice. J. Biol.

Chem. 277, 23781–23787.

BIOGRAPHY

Denis Corbeil is a Group Leader at the University of Dresden. His

research interests are in the cell biology of stem cells, with a focus on

prominin/CD133. He holds a Ph.D. from the University of Montreal

and received postdoctoral training in the laboratory of W.B. Huttner,

where he participated in the molecular cloning of TPST.

Wieland B. Huttner is a Professor of Neurobiology and Director at

MPI-CBG in Dresden. His group made seminal contributions on

protein tyrosine sulfation, including the identiﬁcation, characteriz-

ation, puriﬁcation, and cloning of TPST. He holds an M.D. from the

University of Hamburg, received postdoctoral training with Nobel

Laureate Paul Greengard at Yale University, and has been pursuing

research on neurosecretory vesicle biogenesis and neurogenesis in the

mammalian central nervous system.

TYROSINE SULFATION 297

Ubiquitin System

Aaron Ciechanover and Michael H. Glickman

Technion – Israel Institute of Technology, Haifa, Israel

In the ubiquitin system, a target substrate is modiﬁed by

ubiquitin or a ubiquitin-like protein. In most cases, proteins are

modiﬁed by multiple moieties of ubiquitin, generating a

polyubiquitin chain. This modiﬁcation leads to their degra-

dation by the 26S proteasome complex. Modiﬁcation by a

single moiety of ubiquitin can target proteins for degradation in

the lysosome/vacuole. Modiﬁcation by ubiquitin-like proteins

serves nonproteolytic functions. Conjugation of ubiquitin is

carried out by a modular cascade of enzymes, speciﬁc to each

substrate. Ubiquitination of cellular proteins is a highly com-

plex, temporally controlled, and tightly regulated process that

targets in a speciﬁc manner thousands of cellular proteins and

plays major roles in a variety of basic pathways, such as cell

division, differentiation, and quality control. Not surprisingly,

aberrations in the system underlie the pathogenesis of many

diseases, certain malignancies, and neurodegenerative dis-

orders. Mechanism-based drugs are currently being developed.

Mechanisms of Ubiquitination

and Degradation

UBIQUITINATION

Degradation of a protein via the ubiquitin–proteasome

pathway involves two discrete and successive steps:

(1) tagging of the substrate by covalent attachment of

multiple ubiquitin molecules, and (2) degradation of the

tagged protein by the 26S proteasome complex with

release of free and reusable ubiquitin. This last process is

mediated by ubiquitin-recycling isopeptidases (deubi-

quitinating enzymes). Conjugation of ubiquitin, a highly

evolutionarily conserved 76 residue polypeptide, to the

protein substrate proceeds via a three-step cascade

mechanism. Initially, the ubiquitin-activating enzyme,

E1, activates ubiquitin in an ATP-requiring reaction

to generate a high-energy thiol ester intermediate,

E1-S, ubiquitin. One of several E2 enzymes (ubiquitin-

carrier proteins or ubiquitin-conjugating enzymes

(UBCs)) transfers the activated ubiquitin from E1, via

an additional high-energy thiol ester intermediate,

E2-S , ubiquitin, to the substrate that is speciﬁcally

bound to a member of the ubiquitin-protein ligase

family, E3. There are a number of different classes of E3

enzymes. For the homologous to the E6-AP C-terminus

(HECT) domain E3s, the ubiquitin is transferred once

again from the E2 enzyme to an active site Cys residue

on the E3, to generate a third high-energy thiol ester

intermediate, ubiquitin-S , E3, prior to its transfer to

the ligase-bound substrate. RING ﬁnger-containing E3s

catalyze direct transfer of the activated ubiquitin moiety

to the E3-bound substrate.

E3s catalyze the last step in the conjugation process:

covalent attachment of ubiquitin to the substrate.

The ubiquitin molecule is generally transferred to an

1-NH

group of an internal lysine residue in the substrate

to generate a covalent isopeptide bond. In some cases

however, ubiquitin is conjugated to the N-terminal amino

group of the substrate. By successively adding activated

ubiquitin moieties to internal lysine residues on the

previously conjugated ubiquitin molecule, a polyubi-

quitin chain is synthesized. The chain is recognized by

the downstream 26S proteasome complex. Thus, E3s

play a key role in the ubiquitin-mediated proteolytic

cascade since they serve as the speciﬁc recognition factors

of the system. In certain cases the ﬁrst ubiquitin moiety is

conjugated to the substrate by one E3, while chain

elongation is catalyzed by a different ligase often termed

E4. Modiﬁcation by a single moiety of ubiquitin is

catalyzed via an identical mechanism and set of enzymes.

The speciﬁc enzymes that catalyze modiﬁcation by

ubiquitin-like proteins are somewhat different, though

they utilize a similar mechanism.

DEGRADATION

Degradation of polyubiquitinated substrates is carried

out by a large protease complex, the 26S proteasome

that does not recognize nonmodiﬁed substrates. In one

established case, that of the polyamine synthesizing

enzyme ornithine decarboxylase (ODC), the protea-

some recognizes and degrades the substrate without

prior ubiquitination. The proteasome is a multicatalytic

protease that degrades polyubiquitinated proteins to

short peptides. It is composed of two subcomplexes: a

20S core particle (CP) that carries the catalytic activity,

and a regulatory 19S regulatory particle (RP). The 20S

CP is a barrel-shaped structure composed of four

stacked rings, two identical outer

-rings and two

identical inner

-rings. The eukaryotic

- and

-rings

are composed each of seven distinct subunits, giving

the 20S complex the general structure of

1–7

1–

1–7

. The catalytic sites are localized to some of the

-subunits. Each extremity of the 20S barrel can be

capped by a 19S RP. One important function of the

19S RP is to recognize ubiquitinated proteins and other

potential substrates of the proteasome. A ubiquitin-

binding subunit of the 19S RP has indeed been

identiﬁed; however, its importance and mode of action

have not been discerned. A second function of the 19S

RP is to open an oriﬁce in the

-ring that will allow

entry of the substrate into the proteolytic chamber.

Also, since a folded protein would not be able to ﬁt

through the narrow proteasomal channel, it is assumed

that the 19S particle unfolds substrates and inserts

them into the 20S CP. Both the channel opening

function and the unfolding of the substrate require

metabolic energy, and indeed, the 19S RP contains six

different ATPase subunits. Following degradation of

the substrate, short peptides derived from the substrate

are released, as well as reusable ubiquitin. Proteasomal

degradation is not always complete. In some cases, the

proteasome, rather than completely destroying its

target, processes the ubiquitinated substrate precisely,

releasing a truncated product. In the case of the NF-

transcriptional regulator, an active subunit (p50 or p52)

is thus released from a longer inactive precursor

(p105 or p100). For a general scheme of the ubiquitin

system and its multiple functions, see Figure 1.

SUBSTRATE RECOGNITION

A major unresolved question is how does the system

achieve its high speciﬁcity and selectivity. Why are

certain proteins extremely stable in the cell, while

others are extremely short-lived? Why are certain

proteins degraded only at a particular time point during

the cell cycle or only following speciﬁc extracellular

stimuli, yet they are stable under most other conditions?

It appears that speciﬁcity of the ubiquitin system is

determined by two distinct and unrelated groups of

proteins: E3s and ancillary proteins. First, within

Targeting, protection,

or activation

Substrate

recognition

Ubiquitin ligation

(multiple cycles)

Peptides

E1 E2 E3

Proteolysis

26S

recycling

Ubiquitin ligation

(single cycle)

Targeting

(e.g., lysosome)

Ubl

Ub activation

Processed product

Conjugation

FIGURE 1 The ubiquitin system for protein modiﬁcation. Ubiquitin (Ub) is activated by the three enzymes E1, E2, and E3, and the active

ubiquitin moiety is transferred to the target substrate (red). Substrates can be either modiﬁed by a polyubiquitin chain (A) and targeted to the 26S

proteasome complex for degradation (or processing), or modiﬁed by a single ubiquitin moiety, monoubiquitinated (B) and targeted to other

organelles such as the lysosome (wherein it is degraded). Ubiquitin-like proteins (Ubl) are activated by E1, E2, and E3, which are similar but not

identical to the enzymes that activate ubiquitin. Modiﬁcation by Ubl (C) serves a variety of nonproteolytic functions such as routing of proteins to

subcellular organelles (e.g., nucleus), protecting them from ubiquitination, or activating them.

300 UBIQUITIN SYSTEM

the ubiquitin system, substrates must be speciﬁcally

recognized by an appropriate E3 as a prerequisite to

their ubiquitination. In most cases however, substrates

are not recognized in a constitutive manner, and in

some cases they are not recognized directly by the E3.

In some instances, the E3 must “be switched on” by

undergoing posttranslational modiﬁcation in order to

yield an active form that recognizes the substrate. In

many other cases, it is the substrate that must undergo

a certain change that renders it susceptible for

recognition. The stability of additional proteins

depends on association with ancillary proteins such as

molecular chaperones that act as recognition elements

in trans and serve as a link to the appropriate ligase.

Others, such as certain transcription factors, have to

dissociate from the speciﬁc DNA sequence to which

they bind in order to be recognized by the system.

Stability of yet other proteins depends on oligomeriza-

tion. Thus, in addition to the E3s themselves, modifying

enzymes (such as kinases), ancillary proteins, or DNA

sequences to which substrates bind also play an

important role in the recognition process.

Functions and Substrates

of the Ubiquitin System

Ubiquitin-mediated proteolysis of a variety of cellular

proteins plays an important role in many basic cellular

processes. Among these are regulation of cell cycle and

division, differentiation and development, involvement

in the cellular response to stress and extracellular

effectors, morphogenesis of neuronal networks, modu-

lation of cell surface receptors, ion channels and the

secretory pathway, DNA repair, transcriptional regu-

lation, transcriptional silencing, long-term memory,

circadian rhythms, regulation of the immune and

inﬂammatory responses, and biogenesis of organelles.

The list of cellular proteins that are targeted by ubiquitin

is growing rapidly. Among them are cell-cycle regulators

such as cyclins, cyclin-dependent kinase inhibitors, and

proteins involved in sister chromatid separation, tumor

suppressors, transcriptional activators, and their inhibi-

tors. Cell surface receptors and endoplasmic reticulum

(ER) proteins are also targeted by the system. Finally,

mutated and denatured/misfolded proteins are recog-

nized speciﬁcally and removed efﬁciently. In this

capacity, the system is a key player in the cellular quality

control and defense mechanisms. The products of the

proteasome can play an important role in the immune

response. In the case of degradation of foreign proteins –

such as those of viral origin – the resulting short

peptides are presented by MHC class I molecules to the

cytotoxic T cell that lyse the presenting cell.

Regulation of the Ubiquitin System

The ubiquitin–proteasome pathway can be regulated at

the level of ubiquitination or at the level of proteasome

activity. Since conjugation and proteasomal degradation

is required for multitude of cellular functions, regulation

must be delicately and speciﬁcally tuned. In a few cases,

general rather than speciﬁc, components of the pathway

can be modulated by physiological signals. For example,

up-regulation of the pathway is observed during massive

degradation of skeletal muscle proteins that occurs

under normal fasting, but also under pathological

conditions such as cancer cachexia, severe sepsis,

metabolic acidosis, or following denervation. In most

cases however, regulation is speciﬁc and the target

substrates are recognized by speciﬁc ligases that bind to

deﬁned motifs. The targeting motif can be a single amino

acid residue (e.g., the N-terminal residue) or a sequence

(the “destruction” box in cyclins) or a domain (such as a

hydrophobic patch) that is not normally exposed. In

other cases the motif is a posttranslational modiﬁcation

such as phosphorylation that is generated in response to

cell needs or external signals. Phosphorylation can occur

either on the substrate or on the ligase.

Ubiquitin-Like Proteins

Both enzymes and substrates of the ubiquitin system

have been found to be modiﬁed by ubiquitin-like

proteins. Modiﬁcation by ubiquitin-like proteins occurs

only once. In the case of enzymes, modiﬁcation affects

their activity. For example, the modiﬁcation by the

ubiquitin-like protein NEDD8 enhances the activity of a

certain class of E3 ligases. Conjugation of NEDD8 to

one of the components of the ligase complex (Cullin)

increases its afﬁnity to other components of the

conjugation machinery. In the case of substrates,

modiﬁcation can affect their availability to the ubiqui-

tination/degradation machinery and consequently cellu-

lar stability. For example, in the case of I

, the

inhibitor of the transcriptional regulator NF-

B, modi-

ﬁcation by SUMO-1 was shown to protect the substrate

from ubiquitination. In a completely different case,

SUMOylation of RanGAP1 targets the protein to its

ﬁnal subcellular destination in the nuclear pore complex.

Ubiquitination and Pathogenesis of

Human Diseases

DISEASES

While inactivation of a major enzyme such as E1 is

obviously lethal, mutations or acquired changes in

UBIQUITIN SYSTEM 301

enzymes or in recognition motifs in substrates that do

not affect vital pathways or that affect the involved

process only partially, may result in a broad array of

diseases. The pathological states associated with the

ubiquitin system can be classiﬁed into two groups: those

that result from (1) loss of function – mutation in a

ubiquitin system enzyme or target substrate that result

in stabilization of certain proteins, and (2) gain of

function – abnormal or accelerated degradation of the

protein target.

Alterations in ubiquitination and deubiquitination

reactions have been directly implicated in the etiology of

many malignancies. In general, cancers can result from

“stabilization” of oncoproteins, or “destabilization” of

tumor suppressor gene products. Some of the natural

substrates for degradation by the proteasome are

oncoproteins that, if not properly removed from the

cell, can promote cancer. For instance, ubiquitin targets

N-myc, c-myc, c-fos, c-jun, Src, and the adenovirus E1A

proteins. Destabilization of tumor suppressor proteins

such as p53 and p27 has also been implicated in the

pathogenesis of malignancies.

In one fascinating case, that of uterine cervical

carcinoma, the level of the tumor suppressor protein

p53 is extremely low. Most of these malignancies are

caused by high-risk strains of the human papilloma-

virus (HPV). Detailed studies have shown that the

suppressor is targeted for ubiquitin-mediated degra-

dation by the virally encoded oncoprotein E6. Degra-

dation is mediated by the native HECT domain E3

enzyme E6-AP, where E6 serves as an ancillary protein

that allows recognition of p53 in trans. E6-AP will not

recognize p53 in the absence of E6. E6 associates with

both the ubiquitin-ligase and the target substrate and

brings them to the necessary proximity that is assumed

to allow catalysis of conjugation to occur. Removal of

the suppressor by the oncoprotein is probably

an important mechanism used by the virus to trans-

form cells.

Accumulation of ubiquitin conjugates and/or

inclusion bodies associated with ubiquitin, proteasome,

and certain disease-characteristic proteins have been

reported in a broad array of chronic neurodegenerative

diseases, such as the neuroﬁbrillary tangles of Alzhei-

mer’s disease (AD), brainstem Lewy bodies (LBs) – the

neuropathological hallmark in Parkinson’s disease

(PD), and nuclear inclusions in CAG repeat expansion

(poly-glutamine expansion) disorders such as occurring

Huntington’s disease. However, in all these cases, a

direct pathogenetic linkage to aberrations in the

ubiquitin system has not been established. One factor

that complicates the establishment of such linkage is

the realization that many of these diseases, such as

Alzheimer’s and Parkinson’s, are not deﬁned clinical

entities, but rather syndromes with different etiologies.

Accumulation of ubiquitin conjugates in Lewy

inclusion bodies in many of these cases may be

secondary, and reﬂects unsuccessful attempts by the

ubiquitin and proteasomal machineries to remove

damaged/abnormal proteins. While the initial hypoth-

esis was that inclusion bodies are generated because of

the inherent tendency of the abnormal proteins to

associate with one another and aggregate, it is now

thought that the process may be more complex and

involves active cellular machineries, including inhi-

bition of the ubiquitin system by the aggregated

proteins. This aggregation of brain proteins into deﬁned

lesions is emerging as a common, but poorly under-

stood mechanistic theme in many sporadic and

hereditary neurodegenerative disorders.

The case of Parkinson’s disease highlights the com-

plexity of the involvement of the ubiquitin system in

the pathogenesis of neurodegeneration. Aberrations in

several proteins such as mutations in

-synuclein,

an important neuronal protein, or in the deubiquitinating

enzyme UCH-L1, have been described that link the

ubiquitin system to the pathogenesis of the disease. One

important player in the pathogenesis of Parkinson’s

disease is Parkin which is a RING-ﬁnger E3. Mutations

in the gene appear to be responsible for the pathogenesis

of autosomal recessive juvenile parkinsonism (AR-JP),

one of the most common familial forms of Parkinson’s

disease. Parkin ubiquitinates and promotes the degra-

dation of several substrates. It is possible that aberration

in the degradation of one of these substrates that leads

to its accumulation is neurotoxic and underlies the

pathogenesis of AR-JP.

The cystic ﬁbrosis gene encodes the CF transmem-

brane regulator (CFTR) that is a chloride channel. Only

a small fraction of the protein matures to the cell surface,

whereas most of it is degraded from the ER by the

ubiquitin system prior to its maturation. One frequent

mutation in the channel is DF508. The mutation leads to

an autosomal recessive inherited multisystem disorder

characterized by chronic obstruction of airways and

severe maldigestion due to exocrine pancreatic dysfunc-

tion. Despite normal ion channel function, CFTR

DF508

does not reach the cell surface at all, and is retained in

the ER from which it is degraded. It is possible that the

rapid and efﬁcient degradation results in complete lack

of cell-surface expression of the DF508 protein, and

therefore contributes to the pathogenesis of the disease.

DRUG DEVELOPMENT

Because of the central role the ubiquitin system plays in

such a broad array of basic cellular processes, develop-

ment of drugs that modulate the activity of the system

may be difﬁcult. Inhibition of enzymes common to the

entire pathway, such as the proteasome, may affect many

processes nonspeciﬁcally, although a narrow window

between beneﬁcial effects and toxicity can be identiﬁed

302

UBIQUITIN SYSTEM