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Truncation of Protein Sequences for
Fast Profile Alignment with Application
to Subcellular Localization
Man-Wai MAK and Wei WANG
The Hong Kong Polytechnic University
Sun-Yuan KUNG
Princeton University
Contents
1.
Introduction
–
–
2.
Speeding Up the Prediction Process
–
–
–
3.
4.
Cell Organelles and Proteins Subcellular Localization
Signal-Based vs. Homology-Based Methods
Predicting Cleaving Site Location
Truncating Profiles vs. Truncating Sequences
Perturbational Discriminant Analysis
Experiments and Results
Conclusions
2
Organelles
•
•
•
Cells have a set of organelles that are specialized for carrying out
one or more vital functions.
Proteins must be transported to the correct organelles of a cell to
properly perform their functions.
Therefore, knowing the subcellular localization is one step towards
understanding the functions of proteins.
3
Proteins and Their Subcellular Location
4
Subcellular Localization Prediction
Two key methods:
1. Signal-based
2. Homology-based
5
Signal-Based Method
Source: S. R. Goodman, Medical Cell Biology, Elsevier, 2008.
Cleavage
site
• The amino acid sequence of a protein contains information about its
organelle destination.
• Typically, the information can be found within a short segment of 20
to 100 amino acids preceding the cleavage site.
• Signal-based methods (e.g. TargetP) can determine the cleavage
site location
6
Homology-Based Method
N-dim alignment
vector
Full-length
Query
Sequence
Align with each
of the training
sequences
1
.
.
.
SVM
classifier
Subcellular
Location
N
S(1)=KNKA···
S(2)=KAKN···
·
·
S(N)=KGLL···
Full-length Training
sequences
Advantage:
• Can predict sequences that do
not have cleavage sites.
Drawback:
• Given a query sequence, we
need to align it with every
training sequence in the training
set, causing long computation
7
time.
Sequences Length Distribution
Cleavage Site
Length distribution of Seq.
SP
Occurrences of Seq.
Ext:
21
mTP
820
Cleavage Site
Mit:
1050
35
cTP Cleavage Site
Chl:
Sequence Length
18
760
• Many sequences are fairly long, thus, aligning the whole sequence will take long
computation time.
• cTP, mTP and SP are under 100 AAs only and contain the most relevant segment.
• Computation saving can be achieved by aligning the signal segments only.
8
Proposed Method: Aligning the Segments
that Contain the Most Relevant Info.
N
Amino Acid Sequence
truncate
…
C
Signal-based Cleavage
Site Predictor
(e.g. TargetP)
Cleavage Site
Homology-based
Method
Subcellular
Location
Truncated
sequence
9
Aligning Profiles Vs. Aligning Sequences
Scheme I : Truncate the profiles
Scheme II : Truncate the sequences
Scheme I
PSIBLAST
Long
profile
Cut
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Cut
short
sequence
PSIBLAST
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Query
Sequence
Scheme II
10
Perturbational Discriminant Analysis
Input and Hilbert Spaces:
Input Space
Hilbert Space
Empirical Space:
Empirical
Space
K ( x1, x)

k ( x)  N
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Perturbational Discriminant Analysis
• The objective of PDA is to find an optimal discriminant function in the
Hilbert space or empirical space:
• The optimal solution (see derivation in paper) in the empirical space is
• ρ represents the noise (uncertainty) level in the measurement. It
also ensures numerical stability of the matrix inverse.
• Ρ = 1 in this work.
12
Perturbational Discriminant Analysis
Example on 2-D Data
3 classes of
2-dim data in
the input
space
Projection onto
the 2-dim PDA
space
RBF kernal
matrix K
Decision
boundaries in
the input
space
13
Perturbational Discriminant Analysis
Application to Sequence Classification
Training
sequences
Training
Profiles
PSI-BLAST
Test
sequence
Test
Profile
PSI-BLAST
Pairwise
Alignment
Align with
Training
Profiles
K
Compute
PDA Para
Compute
PDA Score
14
Perturbational Discriminant Analysis
Application to Multi-Class Problems
1-vs-Rest PDA Classifier:
MAXNET
f1 ( x )
fC (x)
f 2 ( x)
x
15
Perturbational Discriminant Analysis
Application to Multi-Class Problems
Cascaded PDA-SVM Classifier:
Test
sequence
Project onto
(C–1)-dim
PDA space
1-vs-rest
SVM
Classifier
Class label
16
Experiments
Materials:
• Eukaryotic sequences extracted from Swiss-Prot 57.5
• Ext, Mit, and Chl contain experimentally determined cleavage sites
• 25% Sequence identity (based on BLASTclust)
Performance Evaluation:
• 5-Fold cross validation
• Prediction accuracy and Matthew’s correlation coefficient (MCC)
17
Comparing Kernel Matrices
Kernel matrix
(Scheme I)
Scheme I
PSIBLAST
Long
profile
Cut
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Cut
short
sequence
PSIBLAST
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Query
Sequence
Scheme II
Kernel matrix
(Scheme II)
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Sensitivity Analysis
Seq
Cut Seq. at p±x
Subcellular Localiation Accuracy (%)
p: gournd-truth cleave site
Subcellular localization
(PairProSVM)
Cyt/Nuc
Ext
Overall
Subcellular
location
• The localization performance
degrades when the cut-off
position drifts away from the
ground-truth cleavage site.
Mit
Chl
• mTP and cTP are more sensitive
to the error of cleavage site
prediction than Ext.
Ground-truth
cleavage site
p-16 p-8
p-2
p p+2 p+16 p+32 p+64
Cut-off Position
19
Csite Prediction ACC(%)
Performance of Cleavage Site Prediction
•
Conditional Random Field
(CRF) is better than
TargetP(Plant) in terms of
predicting the cleavage sites
of signal peptide (Ext) but is
worse than
TargetP(Nonplant).
•
CRF is slightly inferior to
TargetP in predicting the
cleavage sites of
mitochondria, but it is
significantly better than
TargetP in predicting the
cleavage site of chloroplasts.
Category
20
Comparing Profile Creation Time
Scheme I
PSIBLAST
Long
profile
Cut
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Cut
short
sequence
PSIBLAST
short
profile
Pairwise
Alignment
Score
Vector
SVM or
KPDA
Subcellular
Location
Query
Sequence
Scheme II
Findings: Profile creation time can be substantially reduced by
truncating the protein sequences at the cleavage sites.
21
Training and Classification Time
1-vs-rest
SVM
Classifier
MAXNET
f1 ( x )
fC (x)
f 2 ( x)
x
Project onto
(C–1)-dim
PDA space
Findings: The training time of 1-vs-rest PDA and Cascaded PDASVM are substantially shorter than that of SVM.
22
Compare with State-of-the-Art Localization Predictors
Query
seq.
Subcellular localization
(SubLoc/TargetP)
Subcellular
location
Cleavage site prediction
(TargetP/CRF)
Subcellular localization
(PairProSVM)
Localization
Accuracy (%)
MCC
Conditional
Random
Fields
Findings: In terms of localization accuracy, the proposed
“Signal+Homology” method performs slightly better than
the signal-based TargetP and is substantially better than
the homology-based SubLoc.
23
Conclusion
• Fast subcellular-localization-prediction can be
achieved by a cascaded fusion of signal-based and
homology-based methods.
• As far as localization accuracy is concerned, it does
not matter whether we truncate the sequences or
truncate the profiles. However, truncating the
sequence can save the profile creation time by 6
folds.
24
Compare with State-of-the-Art Localization Predictors
Query
seq.
Subcellular localization
(SubLoc/TargetP)
Subcellular
location
Cleavage site prediction
(TargetP/CRF)
Subcellular localization
(PairProSVM)
25
Performance of Cascaded Fusion
Time (hr.)
Time
Subcellular
localization
accuracy
Fulllength
Seq.
Seq. with
Csite
predicted
by
TargetP(P)
Seq. with
Csite
predicted
by
TargetP(N)
Acc
(%)
•
The computation time for
full-length profile
alignment is a striking 116
hours
•
Our method not only
leads to nearly a 20 folds
reduction in computation
time but also boosts the
prediction performance.
Seq. with
Csite
predicted
by CRF
26
Fusion of Signal- and Homology-Based
Methods
1) Cleavage site detection. The cleavage site (if any) of a
query sequence is determined by a signal-based method.
2) Pre-sequence selection. The pre-sequence of the query is
obtained by selecting from the N-terminal up to the cleavage
site.
3) Pairwise alignment. The pre-sequence is aligned with each
of the training pre-sequences to form an N-dim vector, which
is fed to a one-vs-rest SVM classifier for prediction.
N
Amino Acid Sequence
truncate
…
C
Signal-based Cleavage
Site Predictor
Cleavage Site
Homology-based
Method
Subcellular
Location
Pre-sequence
27
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Perturbational Discriminant Analysis
Spectral Space:
Define the kernel matrix
K can be factorized via spectral decomposition into
Empirical Space
K ( x1, x)
Spectral Space

e ( x )  N
28