Lawrence Jk Wee

Calpains belong to a family of calcium-dependent cysteine proteases which are implicated in a myriad of pathologies such as cancer and neurodegeneration. Despite extensive experimental studies on these proteases, our knowledge of the calpain degradome is still limited. Using a dataset of 341 unique, experimentally verified calpain cleavage sites, we conducted extensive sequence analyses and discovered novel residue propensities in the region flanking the cleavage site which could be modeled for prediction using machine learning algorithms. We have developed a series of computational models incorporating support vector machines and Bayes Feature Extraction for the prediction of calpain cleavage sites. The best models achieved AROC and accuracy scores ranging from 0.79 to 0.93 and 71% to 86% respectively when tested on independent test sets. We predicted calpain cleavage sites on proteins from the receptor tyrosine kinase family and discovered potential sites of cleavage at critical regulatory domains. The results suggest a novel role of calpains as a direct regulator of receptor tyrosine kinase activity in cell survival and cell death pathways.

Granzyme B is a serine protease which cleaves at unique tetrapeptide sequences. It is involved in several signaling cross-talks with caspases and functions as a pivotal mediator in a broad range of cellular processes such as apoptosis and inflammation. The granzyme B degradome constitutes proteins from a myriad of functional classes with many more expected to be discovered. However, the experimental discovery and validation of bona fide granzyme B substrates require time consuming and laborious efforts. As such, computational methods for the prediction of substrates would be immensely helpful. We have compiled a dataset of 580 experimentally verified granzyme B cleavage sites and found distinctive patterns of residue conservation and position-specific residue propensities which could be useful for in silico prediction using machine learning algorithms. We trained a series of support vector machines (SVM) classifiers employing Bayes Feature Extraction to predict cleavage sites using sequence windows of diverse lengths and compositions. The SVM classifiers achieved accuracy and AROC scores between 71.00% to 86.50% and 0.78 to 0.94 respectively on independent test sets. We have applied our prediction method on the Chikungunya viral proteome and identified several regulatory domains of viral proteins to be potential sites of granzyme B cleavage, suggesting direct antiviral activity of granzyme B during host-viral innate immune responses. We have compiled a comprehensive dataset of granzyme B cleavage sites and developed an accurate SVM-based prediction method utilizing Bayes Feature Extraction to identify novel substrates of granzyme B in silico. The prediction server is available online, together with reference datasets and supplementary materials.

The identification of B-cell epitopes on antigens has been a subject of intense research as the knowledge of these markers has great implications for the development of peptide-based diagnostics, therapeutics and vaccines. As experimental approaches are often laborious and time consuming, in silico methods for prediction of these immunogenic regions are critical. Such efforts, however, have been significantly hindered by high variability in the length and composition of the epitope sequences, making naïve modeling methods difficult to apply. We analyzed two benchmark datasets and found that linear B-cell epitopes possess distinctive residue conservation and position-specific residue propensities which could be exploited for epitope discrimination in silico. We developed a support vector machines (SVM) prediction model employing Bayes Feature Extraction to predict linear B-cell epitopes of diverse lengths (12- to 20-mers). The best SVM classifier achieved an accuracy of 74.50% and AROC of 0.84 on an independent test set and was shown to outperform existing linear B-cell epitope prediction algorithms. In addition, we applied our model to a dataset of antigenic proteins with experimentally-verified epitopes and found it to be generally effective for discriminating the epitopes from non-epitopes. We developed a SVM prediction model utilizing Bayes Feature Extraction and showed that it was effective in discriminating epitopes from non-epitopes in benchmark datasets and annotated antigenic proteins. A web server for predicting linear B-cell epitopes was developed and is available, together with supplementary materials, at http://www.immunopred.org/bayesb/index.html.

Background: Caspases belong to a class of cysteine proteases which function as critical effectors in cellular processes such as apoptosis and inflammation by cleaving substrates immediately after unique tetrapeptide sites. With hundreds of reported substrates and many more expected to be discovered, the elucidation of the caspase degradome will be an important milestone in the study of these proteases in human health and disease. Several computational methods for predicting caspase cleavage sites have been developed recently for identifying potential substrates. However, as most of these methods are based primarily on the detection of the tetrapeptide cleavage sites - a factor necessary but not sufficient for predicting in vivo substrate cleavage - prediction outcomes will inevitably include many false positives. Results: In this paper, we show that structural factors such as the presence of disorder and solvent exposure in the vicinity of the cleavage site are important and can be used to enhance results from cleavage site prediction. We constructed a two-step model incorporating cleavage site prediction and these factors to predict caspase substrates. Sequences are first predicted for cleavage sites using CASVM or GraBCas. Predicted cleavage sites are then scored, ranked and filtered against a cut-off based on their propensities for locating in disordered and solvent exposed regions. Using an independent dataset of caspase substrates, the model was shown to achieve greater positive predictive values compared to CASVM or GraBCas alone, and was able to reduce the false positives pool by up to 13% and 53% respectively while retaining all true positives. We applied our prediction model on the family of receptor tyrosine kinases (RTKs) and highlighted several members as potential caspase targets. The results suggest that RTKs may be generally regulated by caspase cleavage and in some cases, promote the induction of apoptotic cell death - a function distinct from their role as transducers of survival and growth signals. Conclusion: As a step towards the prediction of in vivo caspase substrates, we have developed an accurate method incorporating cleavage site prediction and structural factors. The multi-factor model augments existing methods and complements experimental efforts to define the caspase degradome on the systems-wide basis.

Background: Caspases belong to a class of cysteine proteases which function as critical effectors in apoptosis and inflammation by cleaving substrates immediately after unique sites. Prediction of such cleavage sites will complement structural and functional studies on substrates cleavage as well as discovery of new substrates. Recently, different computational methods have been developed to predict the cleavage sites of caspase substrates with varying degrees of success. As the support vector machines (SVM) algorithm has been shown to be useful in several biological classification problems, we have implemented an SVM-based method to investigate its applicability to this domain. Results: A set of unique caspase substrates cleavage sites were obtained from literature and used for evaluating the SVM method. Datasets containing (i) the tetrapeptide cleavage sites, (ii) the tetrapeptide cleavage sites, augmented by two adjacent residues, P₁' and P₂' amino acids and (iii) the tetrapeptide cleavage sites with ten additional upstream and downstream flanking sequences (where available) were tested. The SVM method achieved an accuracy ranging from 81.25% to 97.92% on independent test sets. The SVM method successfully predicted the cleavage of a novel caspase substrate and its mutants. Conclusion: This study presents an SVM approach for predicting caspase substrate cleavage sites based on the cleavage sites and the downstream and upstream flanking sequences. The method shows an improvement over existing methods and may be useful for predicting hitherto undiscovered cleavage sites.