Publications -- Jin Liu

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/31807

This collection is limited to articles published under the terms of a creative commons license or other open access publishing agreement since 2016. It is not intended as a complete list of the author's works.

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Identification of Family-Specific Features in Cas9 and Cas12 Proteins: A Machine Learning Approach Using Complete Protein Feature Spectrum
(Cold Spring Harbor Laboratory, 2024-02-08) Madugula, Sita S.; Pujar, Pranav; Bharani, Nammi; Wang, Shouyi; Jayasinghe-Arachchige, Vindi M.; Pham, Tyler; Mashburn, Dominic; Artilis, Maria; Liu, Jin
The recent development of CRISPR-Cas technology holds promise to correct gene-level defects for genetic diseases. The key element of the CRISPR-Cas system is the Cas protein, a nuclease that can edit the gene of interest assisted by guide RNA. However, these Cas proteins suffer from inherent limitations like large size, low cleavage efficiency, and off-target effects, hindering their widespread application as a gene editing tool. Therefore, there is a need to identify novel Cas proteins with improved editing properties, for which it is necessary to understand the underlying features governing the Cas families. In the current study, we aim to elucidate the unique protein attributes associated with Cas9 and Cas12 families and identify the features that distinguish each family from the other. Here, we built Random Forest (RF) binary classifiers to distinguish Cas12 and Cas9 proteins from non-Cas proteins, respectively, using the complete protein feature spectrum (13,495 features) encoding various physiochemical, topological, constitutional, and coevolutionary information of Cas proteins. Furthermore, we built multiclass RF classifiers differentiating Cas9, Cas12, and Non-Cas proteins. All the models were evaluated rigorously on the test and independent datasets. The Cas12 and Cas9 binary models achieved a high overall accuracy of 95% and 97% on their respective independent datasets, while the multiclass classifier achieved a high F1 score of 0.97. We observed that Quasi-sequence-order descriptors like Schneider-lag descriptors and Composition descriptors like charge, volume, and polarizability are essential for the Cas12 family. More interestingly, we discovered that Amino Acid Composition descriptors, especially the Tripeptide Composition (TPC) descriptors, are important for the Cas9 family. Four of the identified important descriptors of Cas9 classification are tripeptides PWN, PYY, HHA, and DHI, which are seen to be conserved across all the Cas9 proteins and were located within different catalytically important domains of the Cas9 protein structure. Among these four tripeptides, tripeptides DHI and HHA are well-known to be involved in the DNA cleavage activity of the Cas9 protein. We therefore propose the the other two tripeptides, PWN and PYY, may also be essential for the Cas9 family. Our identified important descriptors enhanced the understanding of the catalytic mechanisms of Cas9 and Cas12 proteins and provide valuable insights into design of novel Cas systems to achieve enhanced gene-editing properties.
Structure and Dynamics of Cas9 HNH Domain Catalytic State
(Springer Nature, 2017-12-08) Zuo, Zhicheng; Liu, Jin
The bacterial CRISPR-Cas9 immune system has been harnessed as a powerful and versatile genome-editing tool and holds immense promise for future therapeutic applications. Despite recent advances in understanding Cas9 structures and its functional mechanism, little is known about the catalytic state of the Cas9 HNH nuclease domain, and identifying how the divalent metal ions affect the HNH domain conformational transition remains elusive. A deeper understanding of Cas9 activation and its cleavage mechanism can enable further optimization of Cas9-based genome-editing specificity and efficiency. Using two distinct molecular dynamics simulation techniques, we have obtained a cross-validated catalytically active state of Cas9 HNH domain primed for cutting the target DNA strand. Moreover, herein we demonstrate the essential roles of the catalytic Mg(2+) for the active state formation and stability. Importantly, we suggest that the derived catalytic conformation of the HNH domain can be exploited for rational engineering of Cas9 variants with enhanced specificity.
Stress and interferon signalling-mediated apoptosis contributes to pleiotropic anticancer responses induced by targeting NGLY1
(Springer Nature, 2018-11-02) Zolekar, Ashwini; Lin, Victor J. T.; Mishra, Nigam M.; Ho, Yin Ying; Hayatshahi, Hamed S.; Parab, Abhishek; Sampat, Rohit; Liao, Xiaoyan; Hoffmann, Peter; Liu, Jin; Emmitte, Kyle A.; Wang, Yu-Chieh
BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.
Allostery: An Overview of Its History, Concepts, Methods, and Applications
(PLOS, 2016-06-02) Liu, Jin; Nussinov, Ruth
The concept of allostery has evolved in the past century. In this Editorial, we briefly overview the history of allostery, from the pre-allostery nomenclature era starting with the Bohr effect (1904) to the birth of allostery by Monod and Jacob (1961). We describe the evolution of the allostery concept, from a conformational change in a two-state model (1965, 1966) to dynamic allostery in the ensemble model (1999); from multi-subunit (1965) proteins to all proteins (2004). We highlight the current available methods to study allostery and their applications in studies of conformational mechanisms, disease, and allosteric drug discovery. We outline the challenges and future directions that we foresee. Altogether, this Editorial narrates the history of this fundamental concept in the life sciences, its significance, methodologies to detect and predict it, and its application in a broad range of living systems.
Rigid Residue Scan Simulations Systematically Reveal Residue Entropic Roles in Protein Allostery
(PLOS, 2016-04-26) Kalescky, Robert; Zhou, Hongyu; Liu, Jin; Tao, Peng
Intra-protein information is transmitted over distances via allosteric processes. This ubiquitous protein process allows for protein function changes due to ligand binding events. Understanding protein allostery is essential to understanding protein functions. In this study, allostery in the second PDZ domain (PDZ2) in the human PTP1E protein is examined as model system to advance a recently developed rigid residue scan method combining with configurational entropy calculation and principal component analysis. The contributions from individual residues to whole-protein dynamics and allostery were systematically assessed via rigid body simulations of both unbound and ligand-bound states of the protein. The entropic contributions of individual residues to whole-protein dynamics were evaluated based on covariance-based correlation analysis of all simulations. The changes of overall protein entropy when individual residues being held rigid support that the rigidity/flexibility equilibrium in protein structure is governed by the La Chatelier's principle of chemical equilibrium. Key residues of PDZ2 allostery were identified with good agreement with NMR studies of the same protein bound to the same peptide. On the other hand, the change of entropic contribution from each residue upon perturbation revealed intrinsic differences among all the residues. The quasi-harmonic and principal component analyses of simulations without rigid residue perturbation showed a coherent allosteric mode from unbound and bound states, respectively. The projection of simulations with rigid residue perturbation onto coherent allosteric modes demonstrated the intrinsic shifting of ensemble distributions supporting the population-shift theory of protein allostery. Overall, the study presented here provides a robust and systematic approach to estimate the contribution of individual residue internal motion to overall protein dynamics and allostery.
Cas9-catalyzed DNA Cleavage Generates Staggered Ends: Evidence from Molecular Dynamics Simulations
(Springer Nature, 2016-11-22) Zuo, Zhicheng; Liu, Jin
The CRISPR-associated endonuclease Cas9 from Streptococcus pyogenes (spCas9) along with a single guide RNA (sgRNA) has emerged as a versatile toolbox for genome editing. Despite recent advances in the mechanism studies on spCas9-sgRNA-mediated double-stranded DNA (dsDNA) recognition and cleavage, it is still unclear how the catalytic Mg(2+) ions induce the conformation changes toward the catalytic active state. It also remains controversial whether Cas9 generates blunt-ended or staggered-ended breaks with overhangs in the DNA. To investigate these issues, here we performed the first all-atom molecular dynamics simulations of the spCas9-sgRNA-dsDNA system with and without Mg(2+) bound. The simulation results showed that binding of two Mg(2+) ions at the RuvC domain active site could lead to structurally and energetically favorable coordination ready for the non-target DNA strand cleavage. Importantly, we demonstrated with our simulations that Cas9-catalyzed DNA cleavage produces 1-bp staggered ends rather than generally assumed blunt ends.

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