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The Cellular

Chamber off
Structures called proteasomes inside cells continuously
destroy proteins. Several common diseases result when
the process works too zealously or not at all
by Alfred L. Goldberg, Stephen J. Elledge and J.Wade Harper

E very minute of every day a scene straight out of an In-

diana Jones movie plays out in all our cells. One sec-
ond a hapless protein is tooling along just trying to
do its job. The next instant it is branded for destruction and
gets sucked into a dark tunnel, where it is quickly cut to pieces.
Unlike Indiana Jones, for the protein there is no escape. Inside
given time. Genes encode how the 20 basic protein subunits,
called amino acids, are assembled into chains of various
combinations. The chains fold into compact coils and loops
to become different kinds of proteins, each with a specific
function determined by its shape and chemistry.
What happens when proteins are no longer needed or fail
the chamber of doom, the protein is stretched out like a me- to fold correctly? For years, scientists presumed that the
dieval prisoner on the rack and fed through a series of enzy- lions share of protein degradation occurs in lysosomes, bags
matic knives that deliver the Death of a Thousand Cuts. A few of digestive enzymes present in most cells of the body. But in
seconds later the remnants emerge from the tunnel, only to be the early 1970s one of us (Goldberg) showed that cells lack-
pounced on and chewed up further by simpler enzymes. ing lysosomes, such as bacteria and immature red blood cells,
One might think that this intracellular drama is insignifi- can nonetheless destroy abnormal proteins rapidly. What is
cant (except, perhaps, to the unfortunate protein). But scien- more, the process requires energy, whereas other degradative
tists in many laboratories, such as our own, are now finding processes do not.
that these molecular abattoirs, called proteasomes (pro- He and his colleagues were able to get the energy-requir-
nounced pro-tee-ah-somes), are crucial players in pathways ing degradation process to work in test tubes, which enabled
that regulate an entire repertory of cellular processes. A typi- several research groups in the late 1970s and throughout the
cal cell in the body has roughly 30,000 proteasomes. When 1980s to discover the enzymes responsible. Eventually, in
they malfunction whether overeagerly gobbling important 1988, two groups one led by Goldberg and the other by
proteins or failing to destroy those that are damaged or im- Martin C. Rechsteiner of the University of Utah found that
properly formed diseases can ensue. Some viruses, such as the proteins are broken down by large, multienzyme com-
the human immunodeficiency virus (HIV), have even devel- plexes that Goldbergs group named proteasomes.
oped the means to manipulate protein degradation by protea- Proteasomes were so named because they contain many
somes for their own ends. Indeed, several of the next-genera- proteases, enzymes that cut proteins into chunks. But protea-
tion drugs to treat cancer and other dire diseases are expected somes are 100 times larger and more complex than other pro-
to consist of chemical compounds that act on proteasomes teases. Once a protein is laid on the doormat of a proteasome,
and the pathways that feed proteins into proteasomes. Sever- it is taken inside the particle and ultimately disassembled like
al biopharmaceutical companies are now studying com- a Tinker Toy into amino acids that can be reassembled later
pounds that inhibit the proteasome pathway; two such poten-
tial drugs are already in clinical trials in humans.
PROTEASOME draws a protein (ribbonlike structure at
left in lower half ) into its maw for destruction by six specif-
Turnover Is Fair Play
ic enzymes, shown here as knives. An average body cell has
thousands of proteasomes, which chop proteins the cell
P roteins are the very fabric of which cells are made. Some wishes to remove into bits of various sizes. The bits are then

proteins also act as enzymes, the molecular workhorses broken down by other enzymes into the basic building
that drive the chemical reactions of life. The types of proteins blocks of proteins amino acids which are eventually recy-
a cell produces depend on which of its genes are active at any cled to make new proteins.

68 Scientific American January 2001 The Cellular Chamber of Doom

Copyright 2000 Scientific American, Inc.

Copyright 2000 Scientific American, Inc.




E1 E2 E3 E2 E3 E2

into other proteins. Most proteins are her tenants are doing something unde- be degraded by proteasomes. Scientists
replaced every few days, even in cells sirable by monitoring what they throw are finding, for example, that clumps of
that themselves divide rarely, such as out in their daily trash. Although cell misfolded proteins accumulate in asso-
those in the liver or nervous system. proteins are usually degraded all the ciation with proteasomes in certain
And different proteins are degraded at way to amino acids, a few fragments nerve cells, or neurons, in the brains of
widely differing rates: some have half- composed of eight to 10 amino acids people with neurodegenerative disorders
lives as short as 20 minutes, whereas are released by proteasomes, captured, such as Parkinsons, Huntingtons and
others in the same cell may last for days and ultimately displayed on the cells Alzheimers diseases. Why the neurons
or weeks. These rates of breakdown can surface, where the immune system can of individuals stricken with these mal-
change drastically according to chang- monitor whether they are normal or adies fail to degrade the abnormal pro-
ing conditions in our bodies. abnormal [see illustration on page 73]. teins is a burgeoning field of research.
At first glance, such continuous de- Indeed, in disease states and in certain
struction of cell constituents appears tissues such as the spleen and lymph In the Belly of the Beast
very wasteful, but it serves a number of nodes, specialized types of proteasomes
essential functions. Degrading a crucial
enzyme or regulatory protein, for exam-
ple, is a common mechanism that cells
termed immunoproteasomes are pro-
duced that enhance the efficiency of this
surveillance mechanism.
F rom a proteins humble perspective,
proteasomes are enormous struc-
tures. Whereas the average protein is
use to slow or stop a biochemical reac- Protein breakdown by proteasomes 40,000 to 80,000 daltons (or 40,000 to
tion. On the other hand, many cellular also serves as a kind of cellular quality- 80,000 times the molecular weight of a
processes are activated by the degrada- control system that prevents the accu- hydrogen atom), most proteasomes
tion of a critical inhibitory protein, just mulation of aberrant and potentially from higher organisms weigh in at a
as water flows out of a bathtub when toxic proteins. Bacterial and mam- whopping two million daltons. In the
you remove the stopper. This rapid malian cells selectively destroy proteins mid-1990s scientists led by Wolfgang
elimination of regulatory proteins is with highly abnormal conformations Baumeister and Robert Huber of the
particularly important in timing the that can arise from mutation, errors in Max Planck Institute for Biochemistry
transitions between the stages of the cy- synthesis or damage. in Martinsried, Germany, used x-ray
cle that drives cell division [see box on The degradation of abnormal pro- diffraction and electron microscopy to
page 72]. teins is important in a number of hu- determine the molecular architecture of
Protein degradation also plays special man genetic diseases. In various heredi- proteasomes. Each one consists of a
roles in the overall regulation of body tary anemias, a mutant gene leads to the tunnellike core particle with one or two
metabolism. In times of need, such as production of abnormal hemoglobin smaller, regulatory particles positioned
malnourishment or disease, the protea- molecules, which do not fold properly at either or both ends like caps. The
some pathway becomes more active in and are rapidly destroyed by protea- core particle is formed by four stacked
our muscles, providing amino acids that somes soon after synthesis. Similarly, rings each composed of seven sub-
can be converted into glucose and cystic fibrosis is caused by a mutation in units surrounding a central channel
burned for energy. This excessive pro- the gene encoding a porelike protein that constitutes a proteasomes digestive
tein breakdown accounts for the muscle that moves chloride across a cells outer tract. The outer two rings appear to act
wasting and weakness seen in starving membrane. Because these mutant chlo- as gates to keep stray proteins from ac-
individuals and those with advanced ride transporters are slightly misshapen, cidentally bumbling into the degrada-
cancer, AIDS and untreated diabetes. proteasomes degrade them before they tion chamber.
Our immune system, in its constant can reach the cell membrane. The sticky Similarly, the regulatory cap parti-
search to eliminate virus-infected or mucus that builds up in the lungs and cles are thought to act as highly selective
cancerous cells, also depends on protea- other organs of people with cystic fibro- gatekeepers to the core particle. These
somes to generate the flags that distin- sis results from the lack of normal chlo- regulatory particles recognize and bind
guish such dangerous cells. In this pro- ride transporters. to proteins targeted for destruction, then

cess, the immune system functions like a Still other diseases could result in part use energy to unfold the proteins and in-
suspicious landlady checking whether from the failure of abnormal proteins to ject them into the core particle, where

70 Scientific American January 2001 The Cellular Chamber of Doom

Copyright 2000 Scientific American, Inc.


a protein to a proteasome for
they are cut to pieces of various sizes. degradation requires three enzymes
Several research groups have been working in concert to tag the ill-fated
able to synthesize or isolate compounds protein with a chain of ubiquitin mole-
cules. The first enzyme (E1) binds to
that selectively inhibit the proteasome
and activates a ubiquitin molecule and
without affecting other cellular en- then hands it off to the second enzyme
zymes, which could cause side effects. (E2), which in turn joins to a third en-
These inhibitors have been crucial tools zyme (E3). E3 enzymes are like socket
allowing scientists to unravel the intrica- wrenches that fit various target proteins
cies of the proteasome pathway. At high using sockets called F-box proteins.
doses, these inhibitors eventually kill When an E3 binds to a protein, the
cells not surprising in light of the ubiquitin molecule carried by the E2 is
many critical roles that proteasomes broken off and transferred to the pro-
play. But interestingly, cancer cells in the tein. The cycle repeats until the protein
is tagged with a chain of ubiquitins.
test tube and in animals appear more
This chain binds to the protea-
susceptible to these lethal effects than some, which allows enzymes
normal cells do. An experimental pro- near the opening of the pro- FRAGMENTS
teasome inhibitor being tested by Mil- teasome to unfold the pro- OF VARIOUS
lennium Pharmaceuticals in Cambridge, tein and push it into the LENGTHS
Mass., has been evaluated for safety in proteasomes chamber,
humans and will be assessed against where other enzymes INDIVIDUAL
chop it to pieces. AMINO ACIDS
several cancers, including multiple mye-
loma, in trials set to begin this winter.
Another of Millenniums proteasome in-
hibitors is in early safety trials in hu- basic outline for how ubiquitin is at- dreds of distinct E3 proteins that recog-
mans as a possible treatment for stroke tached to a protein has come from nize information in the amino acid se-
and myocardial infarction. Avram Hershko and Aaron Ciechan- quences of other proteins that make
over of the Technion-Israel Institute of them targets for ubiquitination. In re-
The Kiss of Death Technology in Haifa, working with Ir- sponse to altered physiological condi-
win A. Rose of the Fox Chase Cancer tions, such as infection or a lack of nu-

T he proteasome does not just ran-

domly pick out proteins to destroy.
Instead a cell points out which proteins
Center in Philadelphia.
The ubiquitination process has sever-
al steps and involves three enzymes,
trients, cells can modify proteins by
adding phosphate groups. Such phos-
phorylation can alter the activity of a
are doomed. Scientists have discovered dubbed E1, E2 and E3 [see illustration protein or its ability to bind to E3s. Pro-
that the vast majority of such proteins above and on opposite page]. The E1 teins that fail to fold or that become
are first tagged with another protein enzyme activates ubiquitin and con- damaged are also recognized by E3s,
called ubiquitin, for its ubiquity among nects it to E2. The third enzyme, E3, which come along and clean up the pro-
many different organisms. With only 76 then facilitates the transfer of the acti- teins by marking them for pickup by the
amino acids, ubiquitin is a relatively vated ubiquitin from the E2 to the pro- proteasome a little like putting them
tiny protein that can be attached to larg- tein. The process repeats until a long out on the curb on garbage day. Many
er proteins in long chains. These poly- chain of ubiquitins dangles off the pro- key cellular processes rely on protein
ubiquitin tails act like postal codes that tein. That chain is then recognized by a stability, and finding out how stability is
speed doomed proteins to proteasomes. proteasome, which draws the protein in. controlled therefore holds the key to
What controls the timing of a pro- The mystery of how a protein is cho- many of biologys secrets.
teins demise is not its actual breakdown sen for ubiquitination revolves around By controlling the stability of crucial
by the proteasome, but the process of the E3 proteins. Recently researchers, proteins, the E3 proteins regulate many
adding the ubiquitin chains, called ubi- including two of us (Elledge and Harp- cellular processes, such as limb develop-
quitination, which requires energy. The er), have discovered that there are hun- ment, the immune response, cell divi- Scientific American January 2001 71

Copyright 2000 Scientific American, Inc.
sion and cell-to-cell communication. dau (VHL) tumor suppressor, an E3 form of Parkinsons disease results from
Even circadian rhythms and flowering that is often mutated in kidney tumors. a mutation in the gene for a type of E3
in plants are dictated by E3 enzymes. VHLs job is to retard cell growth by enzyme that can cause proteins to build
What is more, several E3s have been limiting the development of blood ves- up in certain brain cells and kill them.
identified as tumor suppressors or onco- sels in tissues; when it is mutated, newly Viruses, which are famous for divert-
genes, tying ubiquitination to the onset formed tumors are able to generate a ing cellular processes, have evolved the
of cancer. rich blood supply and grow rapidly. Sci- means to hijack the process of ubiquitin-
A case in point is the Von Hippel Lin- entists have now found that an inherited ation and protein degradation for their

Why Cell Division Depends on Protein Death

O ne of the best examples of why a cells ability to break identified more than 217 such socket heads, called F-box
down proteins is important for its life and growth proteins, in the nematode worm Caenorhabditis elegans;
comes from studying cell division in Saccharomyces cerevisi- several dozen have been found in human cells so far, and
ae, the common bakers yeast. Before a yeast cell or even a the count is rising.
human cell divides,it must first copy its DNA. And to begin The SCF complex uses its specific set of socket heads to
DNA synthesis, a cell needs to activate a particular class of recognize proteins that should be broken down by the pro-
proteins called S-phase Cdks, which are composed of two teasome. Indeed, cells choose which proteins to degrade by
proteins, a cyclin and a Cdk subunit. adding a phosphate group to them so that they bind to the
S-phase Cdks are normally inactive because they are bound F-box proteins of the SCF. The SCF also serves as a go-be-
to inhibitory proteins (called CKIs) that were made during tween to bring such ill-fated proteins together with the en-
the previous cell division. To activate the S-phase Cdks, a cell zymes that add the ubiquitin death tag.
must get rid of the inhibitory proteins by sending them to a The variety of SCF complexes gives a cell exquisite control
proteasome to be degraded (below). over which types of proteins and how much of each one
Targeting the inhibitory proteins for destruction by a pro- it has on hand at any given time. Proteins regulated by SCF
teasome requires tagging the proteins with a death signal complexes include those that promote or inhibit the cell divi-
called ubiquitin (Ub). This tagging process is normally tightly sion cycle and those that turn on genes. S.J.E.and J.W.H.
regulated; when it goes awry, cells divide uncon-
trollably and some types of cancer can result. INHIBITED
Cells govern the ubiquitination process
and hence the breakdown of proteins that reg- Cdk
ulate cell division using a complex of mole-
Cyclin 1 Enzymes tag inhibitor
cules called the SCF. This complex, which be- Ub for destruction
longs to a group of molecules called the E3s,
acts like a socket wrench with interchangeable
heads that can be swapped to bind only to Cdk
CKI Ub Ub Ub
proteins with particular shapes [see illustrations Cyclin
below and on pages 70 and 71]. Scientists have
2 Tagged inhibitor
enters proteasome and
3 With inhibitor is broken into fragments
MITOSIS gone, S-phase
(cell div on) Cdk is free to CKI Ub Ub Ub
prompt DNA


Cdk Ub


A SY s e)
N T H E SIS (S p ha

72 Scientific American January 2001 The Cellular Chamber of Doom

Copyright 2000 Scientific American, Inc.
IMMUNE SYSTEM relies on spe-
cialized proteasomes called im-
munoproteasomes to help it distin-
guish healthy cells from cancerous IMMUNE CELL
ones or those that have been infect- SURFACE
ed by viruses. In the example shown
at the right, a viral protein is tagged IMMUNO-
with ubiquitin for destruction by
the immunoproteasome. Bits of the PROTEIN
viral protein that are between eight FRAGMENTS
and 10 amino acids in length then
enter the endoplasmic reticulum,
where they are loaded onto newly
formed, forklike molecules called
the major histocompatibility com- ENDOPLASMIC MHC CLASS I
class I molecules are transported APPARATUS
through the Golgi complex and float
to the cell surface, they take along
the viral protein bits. Immune cells
called cytotoxic T cells recognize the
bits of virus embedded in the MHC
class I molecules on the cell surface

as foreign and kill the infected cell.

own nefarious purposes. Human papil- essary for the virus to infect cells but of the proteasome to be destroyed.
lomaviruses (HPVs), which can cause which interferes with the production of New discoveries about the importance
genital warts or cervical or anal cancer, more viruses later on. CD4 acts as a of E3s in disease are rapidly emerging,
are examples. The transformation to docking site for HIV to enter the T cells and these enzymes are likely to be tar-
cancerous growth is usually blocked by of the immune system; it binds to the gets for drug development in the future.
the defense protein p53, one of the gp160 protein that protrudes from the Because each E3 is responsible for the de-
bodys tumor suppressor proteins. surface of the virus. But when HIV struction of a small number of proteins,
HPVs use a trick to circumvent this cel- starts attempting to replicate in the specific inhibitors of E3s should be high-
lular defense system: they make a pro- newly infected cells, CD4 can present ly specific drugs with few side effects.
tein that binds simultaneously to both a problem: it adheres to freshly made The recent identification of large families
p53 and an E3 enzyme. This binding gp160 proteins, keeping them from as- of E3 enzymes have opened up whole
leads to the ubiquitination of p53, sembling with other viral proteins into new avenues for drug discovery. These
which destines p53 to be sliced and new viruses. To circumvent this obsta- are exciting developments that promise
diced to obliteration by the enzymatic cle, HIV has evolved a protein called to enrich the understanding of diverse
Ginsu knives of the proteasome. The Vpu that puts CD4 on the fast track to regulatory phenomena and human biol-
defenseless cells are then more likely to oblivion. Vpu binds to both CD4 and ogy. The more we learn about protea-
become cancers. a complex containing an E3 enzyme, somes and the ubiquitination selection
HIV uses a similar ploy to destroy the causing CD4 to become ubiquitinated machinery, the more we appreciate how
cell-surface protein CD4, which is nec- and then dropped down the chute much of life is linked to protein death. SA

The Authors Further Information

ALFRED L. GOLDBERG, STEPHEN J. ELLEDGE and J. WADE HARPER How the Cyclin Became a Cyclin: Regulated Prote-
have built their careers on studying protein degradation and its role in dis- olysis in the Cell Cycle. Deanna M. Koepp, J. Wade
ease. Goldberg is a professor of cell biology at Harvard Medical School, Harper and Stephen J. Elledge in Cell, Vol. 97, No. 4,
where he received his Ph.D. in 1968 and where he has spent most of his aca- pages 431434; May 14, 1999.
demic life. He has consulted widely for industry; among his honors are the Intricacies of the Proteasome. Stu Borman in Chemi-
1998 Novartis-Drew Award for Biomedical Research. Elledge is the Robert cal and Engineering News, Vol. 78, No. 12, pages 4347;
A. Welch Professor of Biochemistry at Baylor College of Medicine and is an March 20, 2000.
investigator for the Howard Hughes Medical Institute. He received his Ph.D. Probing the Proteasome Pathway. Alfred L. Goldberg
from the Massachusetts Institute of Technology in 1983 and was awarded in Nature Biotechnology, Vol. 18, No. 5, pages 494496;
the Michael E. DeBakey Award for Research Excellence in 1994. Harper is a May 18, 2000.
professor in Baylors department of biochemistry and molecular biology and For more information on the history of studies of the pro-
its department of molecular physiology and biophysics. He received his Ph.D. teasome pathway, visit the Albert and Mary Lasker Foun-
from the Georgia Institute of Technology in 1984 and was granted the Vallee dation Web site at
Visiting Professorship at the University of Oxford in 2000. citation1.html Scientific American January 2001 73

Copyright 2000 Scientific American, Inc.