Transcript Protein Degradation at Lysosome

Protein Targeting and Degradation
David Shiuan
Department of Life Science
Institute of Biotechnology
Interdisciplinary Program of Bioinformatics
National Dong Hwa University
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Protein Targeting and Degradation
• Targeting mechanism involved a peptide signal
sequence
• SRP (signal recognition particle) – move to ER then
Gorgi  lysosome, plasma membrane, transport
vesicles
• Amino-terminal sequence – mitochondria,
chloroplast, bacterial export
• Internal signal sequence – nucleus proteins
• Degradation – ubiquitin-dependent proteolysis at
proteasome
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Directing eukaryotic proteins with signals to ER
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Translocation to ER directed by amino-terminal sequence
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Glycosylation plays a key role in protein targeting
In the ER leumen, signal sequence is removed, protein are
folded, disulfide bond formed and many are glycosylated
A donor molecule brings
the oligo to Asn
Tunicamycin mimics UDP-acetylglucosamine
and block the first of oligo synthesis
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Synthesis of the core oligosaccharide of glycoprotein
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Pathway taken by proteins destined for lysosomes,
the plasma membrane, or secretion
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Phosphorylation of mannose residues on lysosome-targeted enzymes
(hydrolase, the best understood) in Golgi  target to lysosome
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Targeting of nucleus proteins
Ribosomal proteins are imported into nucleus and assembled
into 60s and 40s. The complete subunits are transported back to
cytosol. Nuclear proteins synthesized in cytosol and imported
via importin into nucleus. NLS - nucleus localization signal
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Sanning EM of nucleus surface, showing
numerous nuclear pores
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Signal sequences that target proteins to different
locations in bacteria
+ charge
hydrophobic
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Model for protein export in bacteria
Soluble chaperon protein SecB;
membrane associated SecA (receptor and translocating ATPase)
SecYEG form translocating complex
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Cells import proteins by receptor-mediated endocytosis
Some Proteins (LDL low density lipoprotein, transferrin, peptide
hormone and circulating proteins) destined for degradation – bind to
endocytic receptors concentrated on the protein lattice : clathrin
Clathrin
unit
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Input Sequence
QUERY (348 aa)
MNGTEGPNFY VPFSNKTGVV
RSPFEAPQYY LAEPWQFSML
AAYMFLLIML GFPINFLTLY
VTVQHKKLRT PLNYILLNLA
VADLFMVFGG FTTTLYTSLH
GYFVFGPTGC NLEGFFATLG
GEIALWSLVV LAIERYVVVC
KPMSNFRFGE NHAIMGVAFT
WVMALACAAP PLVGWSRYIP
EGMQCSCGID YYTPHEETNN
ESFVIYMFVV HFIIPLIVIF
FCYGQLVFTV KEAAAQQQES
ATTQKAEKEV TRMVIIMVIA
FLICWLPYAG VAFYIFTHQG
SDFGPIFMTI PAFFAKTSAV
YNPVIYIMMN KQFRNCMVTT
LCCGKNPLGD DEASTTVSKT
ETSQVAPA
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TargetP
• TargetP 1.1 predicts the subcellular location of
eukaryotic proteins
• The location assignment is based on the predicted
presence of any of the N-terminal presequences:
1. Chloroplast transit peptide (cTP)
2. Mitochondrial targeting peptide (mTP)
3. Secretory pathway signal peptide (SP)
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TargetP predictions of unannotated
A. thaliana and H. sapiens sequences
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Bovine cytochrome oxidase
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Bovine cytochrome oxidase
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The Death of Proteins
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Proteins Degradation Rates
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Death by Proteases
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Activation by Proteolysis
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Ubiquitin
2004 Nobel Prize in Chemistry
• Cells are continually building proteins, using them
for a single task, and then discarding them.
• Signaling or controlling proteins (eg. transcription
regulators and the cyclins) - lead very brief lives,
carrying their messages and then being thrown away.
• Specialized enzymes - built just when they are
needed, allowing cells to keep up with their minuteby-minute synthetic needs.
• The approach may seem wasteful, but it allows each
cell to respond quickly to constantly changing
requirements.
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Ubiquitin
2004 Nobel Prize in Chemistry
• The small protein ubiquitin plays a central role.
Ubiquitin attached to obsolete proteins to
dissemble.
• Ubiquitin is found in all eukaryotic cells and in
cells throughout your body.
• The Nobel Prize in Chemistry 2004 was awarded to the
Aaron Ciechanover, Avram Hershko , Irwin
Rose who discovered its essential function in 1980.
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Human Ubiquitin NMR Structure
mqifvktltg ktitleveps dtienvkaki qdkegippdq qrlifagkql
edgrtlsdyn iqkestlhlv lrlrggakkr kkksyttpkk
nkhkrkkvkl avlkyykvde ngkisrlrre cpsdecgagv
fmashfdrhy cgkccltycf nkpedk
156 aa, 8.5 kD
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Ubiquitin Action
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Ubiquitin is attached to a protein by 3 steps
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Eukaryotic Proteosome
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Protein Degradation at Lysosome
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SUMO
(Small Ubiquitin-like Modifier)
• SUMO is member of ubiquitin and ubiquitin like superfamily
• Most SUMO proteins contain tetrapeptide motif
B-K-x-D/E (B: a hydrophobic residue, K: lysine
and D/E: an acidic residue)
• Substrate specificity is derived from ubc9 and
the respective substrate motif
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SUMO and Ubiquitin
• Alignments of ubiquitin and SUMO-1 indicate
only 18% identical in amino acid sequence
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These two proteins have remarkably similar
secondary and tertiary structures
• Unlike the ubiquitin system, which primarily
targets substrate proteins to the proteasome,
SUMO-1 conjugation has diverse cellular
functions
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SUMO
(Small Ubiquitin-like Modifier)
• Unlike ubiquitin modification which targets
proteins for degradation, SUMOylation
1. increases a protein's lifetime.
2. change a protein's location in the cell
• SUMO modification of proteins has many
functions. Among the most frequent and best
studied are protein stability, nuclear-cytosolic
transport, transcriptional regulation (mostly
transcriptional repression).
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SUMO
(Small Ubiquitin-like Modifier)
Human SUMO1: 101 aa; 11.6 kD kD; PDB1A5R
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李 水 龍 (Steven Shoei-Lung Li)
高雄醫學大學醫學研究所 講座教授
Daxx is a shuttle protein participating in biological functions on
various subcellular localizations. Human Fas death domainassociated protein (Daxx) is a 740-amino acids protein mainly
localized in nucleus. It functioned as a transcriptional repressor
when associated with chromatin in nucleus.
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