Download Protein Biosynthesis

Cleavage of a Signal
Sequence
Biosynthesis and Directing secretory proteins to ER
2: Synthesis of
signal peptide
1: Ribosome binds to
the initiation codon
Cytosol
SRP=Signal Recognition
Particle
5: SRP dissociates and recycled and
accompanied by the hydrolysis of GTP.
3: SRP binds to the
ribosome and
halts elongation.
4: The ribosome-SRP complex is bound by the SRP
receptor and GTP on the ER. The peptide, coupled to
peptide translocation complex, inserts into the ER lumen.
7: The signal sequence
is cleaved by signal
peptidase.
Signal sequences targeting different locations
Characteristics of signal sequences:
1. 13-36 amino acid residues
2. 10-15 hydrophobic amino acid residues
3. one or two basic amino acid residues (Lys or Arg) preceding the
hydrophobic sequence
4. cleavage site: Gly or Ala (small side chains)
Protein Targeting to Mitochondria, Chloroplasts and Nuclei
Mitochondria
Chloroplasts
A signal sequence is cleaved off
in mitochondria and chloroplasts
Nuclei
Nuclear localization sequence
(NLS) is not cleaved off.
Internal Peptide Bond
Cleavage
Activation of Proteases
1. After trypsinogen enters the small
intestine, it is converted into its active
form, trypsin by enteropeptidase.
2. Now trypsin hydrolyzes more
trypsinogen and starts to hydrolyze
chymotrpsinogen to active their forms.
Activation of Insulin
1. Insulin is initially synthesized as
preproinsulin.
2. After its assembly in the ER,
preproinsulin is processed into
proinsulin after signal peptide cleavage.
3. Proinsulin undergoes maturation
through the action of several proteases.
4. The remaining polypeptides (51 amino
acids in total), the B- and A- chains
bound together by disulfide bond, are
the active insulin.
Protein Splicing
The Protein Splicing in Gene Expression
The intervening sequence is
transcribed into mRNA and
translated into a nonfunctional
protein precursor.
This precursor undergoes a selfcatalyzed rearrangement in which the
intervening polypeptide segment,
known as the intein, is excised.
The flanking protein segments, the
exteins, are concomitantly joined to
yield the mature protein.
Characteristics of Protein Splicing
1. Protein splicing is catalyzed entirely by amino acid residues
contained in the intein.
2. Protein splicing is an intra-molecular process.
3. Protein splicing requires no coenzymes or sources of
metabolic energy.
4. Protein splicing involves bond rearrangements rather than
bond cleavage, followed by resynthesis.
Conserved Elements in a Typical Intein
A highly conserved amino acid residue is
Asn at the intein C-terminus which can
cyclize, leading to the cleavage of the
intein-C-extein bond.
The C-terminal residue at either splice junction is always an amino acid with a thiol
or hydroxyl side chain, suggesting ester intermediates produced by N-S or N-O acyl
rearrangements.
The Mechanism for Protein Splicing
Formation of a linear ester intermediate by N-O acyl
rearrangement involving the nucleophilic amino acid residue
at the N-terminal junction.
Formation of a branched ester intermediate by the
attack of the nucleophilic residue at the C-terminal
splice junction on the linear ester intermediate.
Cyclization of the Asn residue
coupled with cleavage of the
branched ester intermediate.
Yield an excised intein with a
C-terminal aminosuccinimide
and the two exteins joined by an
ester bond.
Rearrangement of the ester linking the
exteins to the more stable amide bond.
Importance of Protein Splicing
1. Important applications in protein engineering: the elucidation of the
splicing steps to modulate the reactions by mutation and to design
proteins that can undergo self-cleavage and protein ligation reactions.
2. The protein splicing elements can be recognized in other forms of
protein autoprocessing, ranging from peptide bond cleavage to
conjugation with nonprotein moieties.
3. Protein splicing has an ancient evolutionary origin. Protein splicing
elements have been found only in unicellular organisms, whereas the
closely related hedgehog proteins have been found only in animals,
where they play a critical regulatory function in early development.
4. Most inteins harbor homing endonucleases, which turn inteins into
infectious elements by mediating horizontal transfer of the intein coding
sequence.
Protein Lipidation
N-Myristorylation
Structure: CH3(CH2)12CO-NH-GXXXXXX---------COOH
Motif: G ─{EDRHPFYW} ─ χ(2) ─ [STAGCNDEF] ─ {P}
Myristoylation is an irreversible covalent reaction linking
the 14-carbon saturated fatty acid to the N-terminal Gly of
many eukaryotic and viral proteins.
Biosynthesis of N-Myristoryl Proteins
Myristate ＋ ATP
Myistoryl-AMP ＋ PPi
Myistoryl-AMP ＋ CoA
Myistoryl-CoA ＋ AMP
Catalyzed by Acyl-CoA Synthetase
Myistoryl-CoA ＋ Enz
Enz-Myistoryl-CoA
Enz-Myistoryl-CoA ＋ Protein
Enz-Myistoryl-Protein ＋
CoA
Enz-Myistoryl-Protein
Enz ＋ Myistoryl-Protein
Enz= Myistoryl-CoA:Protein N-Myristoryltransferase (NMT)
Importance of Myristoylation
1. The myristate moiety participates in protein subcellular localization by
facilitating protein-membrane interactions as well as protein-protein
interactions.
2. Myristoylated proteins are crucial components of a wide variety of functions,
including many signaling pathways, oncogenesis or viral replication.
3. Initially, myristoylation was described as a co-translational reaction that
occurs after the removal of the initiator Met. It is now established that
myristoylation can also occur post-translationally in apoptotic cells.
4. During apoptosis hundreds of proteins are cleaved by caspases and in many
cases this cleavage exposes an N-terminal Gly within a cryptic
myristoylation consensus sequence, which can be myristoylated.
Co- and Post-translational Attachment of
Myristate to Proteins
Co-translational myristoylation:
following removal of the initiator
Met, the exposed N-terminal Gly
is myristoylated.
Post-translational myristoylation:
following cleavage of a cryptic
myristoylation site by caspase
cleavage, the exposed N-terminl
Gly is myristoylated.
Myristoylation of Proteins During
Apoptosis
Activation of the extrinsic pathway begins
with binding of a death ligand (FasL) to its
corresponding death receptor (Fas).
Subsequent binding of adaptor
proteins leads to the formation of the
death inducing signalling complex
(DISC) and activation of caspase-8.
Caspase-8 cleaves Bid, which
is then myristoylated by
NMT at an N-terminally Gly
of the C-terminal fragment
(ctBid).
The ctBid is essential for
translocation to the
mitochondria and
progression of apoptosis by
the release of cytochrome c.
Gelsolin, b-Actin and PAK2 are all cleaved by
caspase-3 to yield ctActin, ctGelsolin and
ctPAK2, which are subsequently myristoylated.
The caspase-truncated products
translocate to their new
respective membrane locales to
affect apoptosis.
Isoprenylation
C
｜
Structure:(C－C＝C－C－)3-4SCH2－CH－CO－OCH3
｜
NH
｜
C ＝O
｜
Polypeptide
Biosynthesis of C-Terminal Isoprenyl Cysteine Methyl Ester
1. Proteins with a terminal residue Leu are modified by an isoprenyltransferase that
transfers from geranylgeranyl pyrophosphate to Cys. Proteins with terminal residues,
Ser, Ala, Met, or Gln are modified by another enzyme that adds farnesyl pyrophosphate
to Cys.
2. Following the attachment of the isoprenyl moietis, the three terminal amino acids are
cleaved by a protease.
3. Finally, an enzyme catalyzes the addition of a methyl group to the newly exposed
carboxyl terminal Cys.
Isoprenyl Proteins and Their Functions
1. Isoprenyl proteins include the ras proteins and many of the other small Gproteins, the γ-subunits of the large G-proteins, some of the nuclear lamins,
the retinal cGMP Phosphodiesterase, and several fungal mating pheromones.
2. Many isoprenyl proteins function in signal transduction processes across the
plasma membrane or in the control of cell division.
3. The increased hydrophobicity of the C-terminus can lead to interactions with
the membrane bilayer that result in membrane association of these proteins.
4. Alternatively, the isoprenyl and methyl groups may be specific targets for
binding by other membrane "receptor" proteins, leading to a specific
alignment of protein partners in signaling pathways.
The Ras Protein
1. The Ras protein is a product of the ras gene, a mutant version
of a normal gene encoding a GDP-binding protein.
2. The normal protein acts in general transductions triggered by
neurotransmitters, hormones, growth factors and other
extracellular signals.
3. The mutant ras gene is found in cancers of the lung, colon or
pancreas. The mutant gene product is responsible for the
uncontrolled division of the cancerous cells.
S-Palmitorylation
︱
Structure: CH3(CH2)14CO-SCys
︱
1. Protein S-palmitoylation is the thioester linkage of long-chain fatty acids to Cys in
proteins.
2. Addition of palmitate to proteins facilitates their membrane interactions and
trafficking, and it modulates protein-protein interactions and enzyme activity.
3. The reversibility of palmitoylation makes it a bilogical mechanism for regulating
protein activity.
4. The regulation of palmitoylation occurs through the actions of acyltransferases and
acylthioesterases. These molecules work in concert with thioesterases to regulate
the palmitoylation status of numerous signaling molecules, ultimately influencing
their function.
5. It is found mainly in the Ras proteins.
Palmitoylation Motifs
I. Dually lipidated proteins
A. Prenylation and S-palmitoylation
Ras GTPase
–GCMSCKCfarn
Rho GTPase
–QNGCINCCfarn/gg
B. N-myristoylation and S-palmitoylation
Gpal (S. cerevisiae)
myr-N-GCTVST–
C. N-palmitoylation and S-palmitoylation
Gs
palm-N-GCLGNSKTE–
II. Cytoplasmic proteins—exclusively palmitoylated
A. N-terminal motifs
1MLCCM–
GAP-43
B. C-terminal motif
Yck2 (S. cerevisiae)
–FFSKLGCC-COOH
C. Cysteine string motifs
SNAP-25b
–83KFCGLCVCPCNKL95–
III. Pleckstrin homology domains
Phospholipase D1
–236PGLNCCGQGR245–
VI. RGS core domains
RGS4
–91FWISCEEYKKI103–
Two Mechanisms of Protein Palmitoylation:
Enzymatic and Nonenzymatic
1. The first mechanism is through the action of an enzyme
generically referred to as protein acyltransferase (PAT).
Genetic strategies in yeast have recently yielded the
identity of two PATs. Thus, it is clear that some
palmitoylation reactions are mediated by enzymes.
2. The second mechanism is nonenyzmatic: spontaneous
autoacylation of a protein in the presence of long-chain
acyl-coenzyme As (CoAs). Autoacylation of two
mitochondrial enzymes is important for their regulation.
Functions of Palmitoylation
1. Similar to other lipid modifications, palmitoylation promotes membrane
association of otherwise soluble proteins.
2. The function of palmitoylation, however, ranges beyond that of a
simple membrane anchor.
3. Trafficking of lipidated proteins from the early secretory pathway to the
plasma membrane is dependent upon palmitoylation in many cases.
4. Modification with fatty acids impacts the lateral distribution of proteins
on the plasma membrane by targeting them to lipid rafts.
5. Palmitoylation also functions in the regulation of protein activity.
6. SCG-10, a microtubule-destabilizing protein, requires palmitoylation
for targeting to Golgi membranes for transport to neuronal growth
cones.
Glypiation
1. The process of adding glycosyl phosphatidyl inositol (GPI) to proteins, which
has been termed glypiation, is carried out by a transamidase that cleaves the
C-terminal peptide and concomitantly transfers the preassembled GPI anchor
to the newly exposed carboxy-terminal amino acid residue to establish an
amide bond between the latter and the ethanolamine moiety of the glycolipid.
2. GPI assembly takes place entirely on the cytoplasmic side of the ER and
followed by its translocation to the lumenal side, where attachment to the
protein takes place.
3. The transamidase reaction is carried out by a multiprotein complex that has as
yet not been isolated in its intact form.
4. The carboxy-terminal signal peptide which is cleaved prior to binding of the
GPI, consisting 15–30 amino acids, has structural similarities to the NH2terminal peptide that functions in general to direct nascent chains into the ER
lumen.
GPI Anchor
The GPI anchor is a complex structure
comprising a phosphoethanolamine
linker, glycan core, and phospholipid
tail.
Membrane-Associated Proteins in a Lipid Bilayer
Containing Lipid Raft Domains
The GPI anchor anchors the
modified protein in the outer
leaflet of the cell membrane.
Sequences Signaling the Addition of GPI Anker
1. Addition involves removal of the sequence to the right of the space and linkage of
the GPI to the residue at the left of the space.
2. The residue to which GPI becomes attached (termed ω) has small side chains (e.g.,
Gly, Ala, Cys, Ser, Asn) as does the amino acid in the ω+2 position (e.g., Gly, Ala).
3. The latter site is followed by a short hydrophilic domain (5–7 residues) and this is
followed by a hydrophobic region (12–20 residues) that extends to the carboxyterminus. The ω+1 position can be filled by any amino acid except Pro or Trp.
Biosynthesis of GPI Anchor
In trypanosomes
In mammalian cells
PI, phophatidylinositol; G, glucosamine;
Ac, acetyl; M, mannose; pE,
Phosphoehanolamine; (acyl), fatty acid
linked to inositol.
Functions of GPI-Anchored Proteins
────────────────────────────────────────────────────────────────
Biological role
Protein
Source
────────────────────────────────────────────────────────────────
enzymes
alkaline phosphatase
mammalian tissues, Schistosoma
5′-nucleotidase
mammalian tissues
dipeptidase
pig and human kidney, sheep lung
cell-cell interaction
LFA-3
human blood cells
PH-20
guinea pig sperm
complement regulation
CD55 (DAF)
human blood cells
CD59
human blood cells
mammalian antigens
Thy-1
mammalian brain and lymphocytes
Qa-2
mouse lymphocytes
CD14
human monocytes
CD52
human lymphocytes
protozoan antigens
VSG
T. brucei
1G7
T. cruzi
procyclin
T. brucei
miscellaneous
scrapie prion protein
hamster brain
CD16b
human neutrophils
folate-binding protein
human epithelial cells
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Difficulties in the Studies of GPI-Proteins
1. The GPI anchor is a structurally complex posttranslational
modification that remains a mystery with respect to its
biological activities.
2. A major obstacle to functional investigations is the difficulty
in producing structurally defined GPI moieties and GPIanchored proteins.
3. Cells generate heterogeneous mixtures of both anchor and
modified protein structures, and chemical synthesis requires
numerous difficult synthetic transformations.
4. The flexibility of glycosidic linkages and the solubility issues
inherent to lipids have rendered GPImodified proteins
difficult to crystallize or to study by NMR.
Cholesterol-Modified Proteins:
Hedgehog (Hh) proteins
1. Hh proteins are the representative of cholesterol-modified
proteins and carry a cholesterol ester at the C terminus of
their signaling domain.
2. They comprise a family of secreted signaling molecules
essential for patterning a variety of structures in animal
embryogenesis.
Biosynthesis of Hh Proteins
1. During biosynthesis, Hh undergoes an autocleavage reaction,
mediated by its carboxyl-terminal domain, that produces a
lipid-modified amino-terminal fragment responsible for all
known Hh signaling activity.
2. Cholesterol is the lipophilic moiety covalently attached to the
amino-terminal signaling domain during autoprocessing and
that the carboxyl-terminal domain acts as an intramolecular
cholesterol transferase.
3. This use of cholesterol to modify embryonic signaling proteins
may account for some of the effects of perturbed cholesterol
biosynthesis on animal development.
Mechanism of Hh Autoprocessing and Attachment of
Cholesterol
The reaction is initiated by formation of a
thio-ester between the thiol side chain of
Cys258 and the carbonyl carbon of Gly257,
an N to S shift.
The activated intermediate then undergoes
a nucleophilic attack by DTT or by a
lipophilic nucleophile (cholesterol),
resulting in cleavage as well as formation
of a covalent adduct at the COOH-terminus
of the NH2-terminal product.
X denotes the attacking nucleophile
Disulfide Bonds
Historical Events on Disulfide Formation
1. Anfinsen’s experiment showed that the reduced RNase
could be oxidized spontaneously in air, but it took hours to
do so. In the cell, the process is more rapid. It takes only
seconds.
2. The discovery of mutations in the gene, dsbA, revealed that
disulfide formation is a catalyzed process.
Thiol-Redox Reactions
This figure shows the 3 thiol-disulfide interconversions between an
enzyme (E) with 2 active Cys and a substrate (S) with 3 Cys.
The commonality of
mechanism is shown for the
three series of reactions.
The mixed disulfide intermediate is formed by
the attack by a depronated thiolate anion.
The central complex
represents one of the six
possible mixed
disulfide intermediates.
Dsb A, Primary Catalyst for Disulfide Formation
1. DsbA is a monomeric 21-kDa enzyme containing a redox active sequence,
Cys-Pro-His-Cys, embedded in the thioredeoxin-like fold.
2. DsbA, with a standard redox potential of about –120 mV, is the strongest
thiol oxidant.
3. The first Cys (Cys30) has a pKa value of 3.5. This Cys is thus entirely in the
thiolate state at physiological pH.
4. 3-D structure shows that this Cys is located at the N-terminus of the active
site helix 1 from which the positive charge of the helix dipole can stabilize
the thiolate.
5. 3-D structure also shows that His32 in the CXXC motif is hydrogen-bonded
to Cys30 in the reduced but not in the oxidized form.
Electron Transfer from Substrate to DsbA and DsbB
1. The protease digestion of DsbA suggested that the
oxidized form is more flexible than the reduced
form.
2. The greater flexibility of the oxidized form can
facilitate the accommodation of its various
substrates, while the rigid nature of the reduced
form can facilitate the release of the oxidized
products.
DsbB is responsible for
maintaining DsbA oxidized.
DsbC, Protein Disulfide Bond Isomerase
1. The slowness of the spontaneously
unscrambling of the scrambled RNase led
Anfinsen to isolate a protein disulfide bond
isomerase (DsbC) which promotes the
rearrangements of nonnative disulfide bonds.
2. The dimeric structure is fundamental for
disulfide bond isomerase and as a chaperon.
3. As a chaperon, DsbC can reactivate
denatured proteins without Cys, and the
active site Cys of DsbC is not required for
the chaperon activity.
4. The recognition of unfolded proteins is in
the uncharged cleft which can switch
between open and closed conformation.
5. The flexibility allows DsbC to adjust the
cleft to accommodate different sizes and
shapes of the binding partner.
DsbC is a V-shaped homodimer (monomer,
23.4 kD) where each arm consists of a Cterminal thioredoxin fold with an active
CXXC motif and an N-terminal dimerization
domain.
DsbD, An Enzyme
Responsible for
Maintaining DsbC in the
Reduced State
DsbD consists of an N-terminal
periplasmic domain (DsbDα), a central
hydrophobic core with 8 transmembrane
segments (DsbDβ) and a C-terminal
thioredoxin-like domain (DsbDγ). Each of
the three domains includes a pair of Cys.
DsbD utilizes the thioredoxin/thioredoxin
reductase system in the cytosol as a source
of reducing equivalents.
The reducing potential is
transferred from thioredoxin
to DsbDβ and then
successively to DsbDγ,
DsbDα, and DsbC.
Protein Ubiquitination
Ubiquitin System
The conjugation of Ub to proteins involves a ATPdependent step in which the last residue of Ub
(Gly76) is joined to a Cys residue of the E1 (Ubactivating) enzyme.
The “activated” Ub moiety is
transferred to a Cys residue in one
of several Ub-conjugating (E2)
enzymes and, from there, through
an isopeptide bond to a Lys residue
of an ultimate acceptor protein.
E2 enzymes function as
subunits of E2-E3 ligase
holoenzyme that can produce
substrate-linked poly-Ub
chains.
Substrate linked poly-Ub chains with specific Ub-Ub
isopeptide bonds mediate either the processive degradation of
a substrate by the 26S proteasome or other metabolic fates.
The Arg/N-End Rule Pathway
1.Primary destabilizing N-terminal residues (Arg, Lys, His, Leu, Phe, Tyr, Trp, and Ile),
are directly recognized by E3-E2 ligase.
2.The N-terminal residues Asp, Glu, Asn, and Gln can be targeted by E3-E2 ligase only
after their N-terminal arginylation by the Ate1 Arg-tRNA-protein transferase (Rtransferase).
3.The destabilizing residues are called secondary or tertiary, depending on the number of
steps (arginylation of Asp and Glu; deamidation/arginylation of Asn and Gln) that
precede the targeting.
4.Specifically, the targeting apparatus of the Arg/N-end rule pathway comprises a physical
complex of the RING-type E3 (called N-recognin) and the HECT-type E3, together with
their cognate E2 enzymes, respectively.
The Ac/N-End Rule Pathway
1.The red arrow indicates the removal of N-terminal Met by Met aminopeptidases (MetAPs).
2.This Met residue is retained if a residue at position 2 is nonpermissive (too large) for
MetAPs. If the retained N-terminal Met or N-terminal Ala, Val, Ser, Thr, and Cys are
followed by acetylation-permissive residues, the above N-terminal residues are usually Nterminally acetylated by Nt-acetylases . The resulting N-degrons are called AcN-degrons.
3.The term “secondary” refers to the necessity of modification (Nt-acetylation) of a
destabilizing N-terminal residue before a protein can be recognized by a cognate Ub ligase.
4.Although second-position Gly or Pro can be made N-terminal by MetAPs, and although
the Doa10 E3 can recognize Nt-acetylated Gly and Pro, few proteins with N-terminal Gly
or Pro are Nt-acetylated.
Functions of Ubiquitination
1. The function of ubiquitination was to cause protein degradation in the 26 S
proteasome, if the linkage to the target protein is via Lys48 on ubiquitin (Ub). Ubmediated degradation of regulatory proteins plays important roles in the control of
numerous processes, including cell-cycle progression, signal transduction,
transcriptional regulation, receptor down-regulation, and endocytosis
2. If the polyubiquitin chains are linked via Lys63, a major function is to assemble
into multiprotein complexes and regulates processes such as endocytosis and
ribosomal protein synthesis.
3. If the process of ubiquitination is mediated by Ub ligases and is reversed by
deubiquitinating enzymes, the phenomenon can be found in the regulation of
innate immune signaling, where both phosphorylation and Lys63-linked
ubiquitination are the critical covalent modifications that launch signaling
pathways activated by innate immune receptors.
4. Monoubiquitylation of specific substrates has specific functions.
5. An individual mammalian genome encodes at least a thousand distinct E3s. One
role of E3 is the initial recognition of a substrate’s degradation signal (degron). .
6. The ubiquitin system has been implicated in the immune response, development,
and programmed cell death.
Protein Sumonylation
Small ubiquitin-related modifier is referred to as
SUMO and protein sumonylation is a
posttranlational process.
The SUMO Conjugation Pathway
1. The enzymes of the SUMO pathway are analogous to those of the Ub pathway with a SUMOactivating enzyme (E1), a SUMO-conjugating enzyme (E2) and one of several SUMO-protein
ligases (E3s).
2. E2 and the E3s both contribute to substrate specificity.
3. The linkage between SUMO and its substrates is an isopeptide bond between the C-terminal
carboxyl group of SUMO and the ε-amino group of Lys in the substrate. The Lys residues
where SUMO becomes attached are in the short consensus sequence ΨKXE, where is Ψ a
large hydrophobic amino acid, generally Ile, Leu, or Val and X is any residue.
4. Sumoylation is a reversible modification, and removal of SUMO is carried out by enzymes of
the Ulp family.
5. Ulps are also required for generating mature SUMO from the SUMO precursor, which
contains a short peptide blocking its C terminus.
6. The enzymes of the SUMO pathway are specific for SUMO and have no role in conjugating
Ub or any of the other Ubls.
The Structures of SUMOs
1. SUMOs are 11 kDa proteins, but they appear larger on SDSPAGE and add 20 kDa to the apparent molecular weight of
most substrates.
2. SUMOs are 20 amino acids longer than Ub, and the extra
residues are found in an N-terminal extension, which is
flexible in solution.
3. The N-terminal extension of yeast SUMO can be entirely
deleted with only modest effects on SUMO function,
indicating that the Ub-like domain is sufficient for
conjugation to many substrates.
Comparison of the Structures of SUMO and
Ubiquitin
SUMOs share 18% sequence identity with Ub, but the folded
structure of the SUMO C-terminal Ub-like domain is virtually
superimposable on that of Ub.
Functions of Sumonylation
1. SUMO modifies many proteins that participate in diverse cellular
processes, including transcriptional regulation, nuclear transport,
maintenance of genome integrity, and signal transduction.
2. Reversible attachment of SUMO is controlled by an enzyme pathway that
is analogous to the ubiquitin pathway but not for protein degradation.
3. The functional consequences of SUMO attachment vary greatly from
substrate to substrate, and in many cases are not understood at the
molecular level.
4. Frequently SUMO alters interactions of substrates with other proteins or
with DNA, but SUMO can also act by blocking ubiquitin attachment sites.
5. An unusual feature of SUMO modification is that, for most substrates,
only a small fraction of the substrate is sumoylated at any given time.