Xin Zhang
@psu.edu
Associate Professor, Department of Biochemistry and Molecular Biology, Department of Chemistry
Penn State University
Scopus Publications
Scholar Citations
Scholar h-index
Scholar i10-index
Scopus Publications
Yanan Huang, Junlin Chen, Chia-Heng Hsiung, Yulong Bai, Zizhu Tan, Songtao Ye, and Xin Zhang
American Society for Cell Biology (ASCB)
The formation of cellular condensates, akin to membraneless organelles, is typically mediated by liquid−liquid phase separation (LLPS), during which proteins and RNA molecules interact with each other via multivalent interactions. Gaining a comprehensive understanding of these interactions holds significance in unraveling the mechanisms underlying condensate formation and the pathology of related diseases. In an attempt toward this end, fluorescence microscopy is often used to examine the colocalization of target proteins/RNAs. However, fluorescence colocalization is inadequate to reliably identify protein interaction due to the diffraction limit of traditional fluorescence microscopy. In this study, we achieve this goal through adopting a novel chemical biology approach via the dimerization-dependent fluorescent proteins (ddFPs). We succeeded in utilizing ddFPs to detect protein interaction during LLPS both in vitro and in living cells. The ddFPs allow us to investigate the interaction between two important LLPS-associated proteins, FUS and TDP-43, as cellular condensates formed. Importantly, we revealed that their interaction was associated with RNA binding upon LLPS, indicating that RNA plays a critical role in mediating interactions between RBPs. More broadly, we envision that utilization of ddFPs would reveal previously unknown protein−protein interaction and uncover their functional roles in the formation and disassembly of biomolecular condensates.
Junbao Ma, Rui Sun, Kaifu Xia, Qiuxuan Xia, Yu Liu, and Xin Zhang
American Chemical Society (ACS)
Xin Zhang and Squire J. Booker
American Chemical Society (ACS)
Baoxing Shen, Lihua Liu, Yubo Huang, Jichun Wu, Huan Feng, Yu Liu, He Huang, and Xin Zhang
Wiley
AbstractMolecular rotor‐based fluorophores (RBFs) activate fluorescence upon increase of micro‐viscosity, thus bearing a broad application promise in many fields. However, it remains a challenge to control how fluorescence of RBFs responds to viscosity changes. Herein, we demonstrate that the formation and regulation of intramolecular hydrogen bonds in the excited state of RBFs could modulate their rotational barrier, leading to a rational control of how their fluorescence can be activated by micro‐viscosity. Based on this strategy, a series of RBFs were developed based on 4‐hydroxybenzylidene‐imidazolinone (HBI) that span a wide range of viscosity sensitivity. Combined with the AggTag method that we previously reported, the varying viscosity sensitivity and emission spectra of these probes enabled a dual‐color imaging strategy that detects both protein oligomers and aggregates during the multistep aggregation process of proteins in live cells. In summary, our work indicates that installing intracellular excited state hydrogen bonds to RBFs allows for a rational control of rotational barrier, thus allow for a fine tune of their viscosity sensitivity. Beyond RBFs, we envision similar strategies can be applied to control the fluorogenic behavior of a large group of fluorophores whose emission is dependent on excited state rotational motion, including aggregation‐induced emission fluorophores.
Xuejun Cheng, Yingming Pu, Songtao Ye, Xiao Xiao, Xin Zhang, and Hongyu Chen
Wiley
AbstractMeasuring the diffusivity of molecules is the first step toward understanding their dependence and controlling diffusion, but the challenge increases with the decrease of molecular size, particularly for non‐fluorescent and non‐reactive molecules such as solvents. Here, the capability to monitor the solvent exchange process within the micropores of silica with millisecond time resolution is demonstrated, by simply embedding a rotor‐based fluorophore (thioflavin T) in colloidal silica nanoparticles. Basically, the silica provides an extreme case of viscous microenvironment, which is affected by the polarity of the solvents. The fluorescence intensity traces can be well fitted to the Fickian diffusion model, allowing analytical solution of the diffusion process, and revealing the diffusion coefficients. The validation experiments, involving the water‐to‐ethanol and ethanol‐to‐water solvent exchange, the comparison of different drying conditions, and the variation in the degree of cross‐linking in silica, confirmed the effectiveness and sensitivity of this method for characterizing diffusion in silica micropores. This work focuses on the method development of measuring diffusivity and the high temporal resolution in tracking solvent exchange dynamics over a short distance (within 165 nm) opens enormous possibilities for further studies.
Huan Feng, Qun Zhao, Nan Zhao, Zhen Liang, Yanan Huang, Xin Zhang, Lihua Zhang, and Yu Liu
Wiley
AbstractIntracellular proteome aggregation is a ubiquitous disease hallmark with its composition associated with pathogenicity. Herein, this work reports on a cell‐permeable photosensitizer (P8, Rose Bengal derivative) for selective photo induced proximity labeling and crosslinking of cellular aggregated proteome. Rose Bengal is identified out of common photosensitizer scaffolds for its unique intrinsic binding affinity to various protein aggregates driven by the hydrophobic effect. Further acetylation permeabilizes Rose Bengal to selectively image, label, and crosslink aggregated proteome in live stressed cells. A combination of photo‐chemical, tandem mass spectrometry, and protein biochemistry characterizations reveals the complexity in photosensitizing pathways (both Type I & II), modification sites and labeling mechanisms. The diverse labeling sites and reaction types result in highly effective enrichment and identification of aggregated proteome. Finally, aggregated proteomics and interaction analyses thereby reveal extensive entangling of proteostasis network components mediated by HSP70 chaperone (HSPA1B) and active participation of autophagy pathway in combating proteasome inhibition. Overall, this work exemplifies the first photo induced proximity labeling and crosslinking method (namely AggID) to profile intracellular aggregated proteome and analyze its interactions.
Jun Wang, Xinwei Ma, Yi Hu, Guanhua Feng, Chunce Guo, Xin Zhang, and Hong Ma
Springer Science and Business Media LLC
AbstractPre-mRNA splicing is crucial for gene expression and depends on the spliceosome and splicing factors. Plant exons have an average size of ~180 nucleotides and typically contain motifs for interactions with spliceosome and splicing factors. Micro exons (<51 nucleotides) are found widely in eukaryotes and in genes for plant development and environmental responses. However, little is known about transcript-specific regulation of splicing in plants and about the regulators for micro exon splicing. Here we report that glycine-rich protein 20 (GRP20) is an RNA-binding protein and required for splicing of ~2,100 genes including those functioning in flower development and/or environmental responses. Specifically, GRP20 is required for micro-exon retention in transcripts of floral homeotic genes; these micro exons are conserved across angiosperms. GRP20 is also important for small-exon (51–100 nucleotides) splicing. In addition, GRP20 is required for flower development. Furthermore, GRP20 binds to poly-purine motifs in micro and small exons and a spliceosome component; both RNA binding and spliceosome interaction are important for flower development and micro-exon retention. Our results provide new insights into the mechanisms of micro-exon retention in flower development.
Cong Liu and Xin Zhang
Elsevier BV
Yulong Bai, Shengnan Zhang, Hui Dong, Yu Liu, Cong Liu, and Xin Zhang
American Chemical Society (ACS)
Protein misfolding and aggregation, a key contributor to the progression of numerous neurodegenerative diseases, results in functional deficiencies and the creation of harmful intermediates. Detailed visualization of this misfolding process is of paramount importance for improving our understanding of disease mechanisms and for the development of potential therapeutic strategies. While in vitro studies using purified proteins have been instrumental in delivering significant insights into protein misfolding, the behavior of these proteins in the complex milieu of living cells often diverges significantly from such simplified environments. Biomedical imaging performed in cell provides cellular-level information with high physiological and pathological relevance, often surpassing the depth of information attainable through in vitro methods. This review highlights a variety of methodologies used to scrutinize protein misfolding within biological systems. This includes optical-based methods, strategies leaning on mass spectrometry, in-cell nuclear magnetic resonance, and cryo-electron microscopy. Recent advancements in these techniques have notably deepened our understanding of protein misfolding processes and the features of the resulting misfolded species within living cells. The progression in these fields promises to catalyze further breakthroughs in our comprehension of neurodegenerative disease mechanisms and potential therapeutic interventions.
Haoran Wang, Qiyao Li, Parvej Alam, Haotian Bai, Vandana Bhalla, Martin R. Bryce, Mingyue Cao, Chao Chen, Sijie Chen, Xirui Chen,et al.
American Chemical Society (ACS)
Light has profoundly impacted modern medicine and healthcare, with numerous luminescent agents and imaging techniques currently being used to assess health and treat diseases. As an emerging concept in luminescence, aggregation-induced emission (AIE) has shown great potential in biological applications due to its advantages in terms of brightness, biocompatibility, photostability, and positive correlation with concentration. This review provides a comprehensive summary of AIE luminogens applied in imaging of biological structure and dynamic physiological processes, disease diagnosis and treatment, and detection and monitoring of specific analytes, followed by representative works. Discussions on critical issues and perspectives on future directions are also included. This review aims to stimulate the interest of researchers from different fields, including chemistry, biology, materials science, medicine, etc., thus promoting the development of AIE in the fields of life and health.
Qianhui Xu, Yeyang Ma, Yunpeng Sun, Dan Li, Xin Zhang, and Cong Liu
Wiley
AbstractProtein amyloid aggregation has been widely observed to occur and plays important roles in both physiological processes and pathological diseases. Remarkably, amyloid aggregates assembled by native proteins gain a variety of different biological activities, which cannot be adopted by the unassembled protein alone. Thus, it is important to investigate the molecular basis of self‐assembly of protein amyloid aggregates and how the aggregated protein structure determines its function. In the review, we firstly introduce our structural knowledge on how different amyloid proteins undergo conformational transition and assemble into amyloid aggregate, with the main focus on amyloid fibril, which is the major species of amyloid aggregate. Then, we elaborate how different structures of amyloid fibrils enable them to fulfill highly diverse functions in either physiological or pathological condition. Furthermore, we discuss the structural polymorph which is a very unique feature of amyloid fibril, and its implication in understanding the structure‐function relationship of amyloid fibrils. Finally, we point out the importance of applying and integrating new approaches for deepening the structure‐function study of amyloid fibrils and highlight the potential of designing amyloid fibril‐based functional bio‐nanomaterials for application.
Baoxing Shen, Kwan Ho Jung, Songtao Ye, Conner A. Hoelzel, Charles H. Wolstenholme, He Huang, Yu Liu, and Xin Zhang
Wiley
AbstractAberrant protein aggregation leads to various human diseases, but little is known about the physical chemical properties of these aggregated proteins in cells. Herein, we developed a boron‐dipyrromethene (BODIPY)‐based HaloTag probe, whose conjugation to HaloTag‐fused proteins allows us to study protein aggregates using both fluorescence intensity and lifetime. Modulation of BODIPY fluorophore reveals key structural features to attain the dual function. The optimized probe exhibits increased fluorescence intensity and elongated fluorescence lifetime in protein aggregates. Fluorescence lifetime imaging using this probe indicates that protein aggregates afford different viscosity in the forms of soluble oligomers and insoluble aggregates in live cells. The strategy presented in this work can be extended to enable a wide class of HaloTag probes that can be used to study a variety of physical properties of protein aggregates, thus helping unravel the pathogenic mechanism and develop therapeutic strategy.
Di Shen, Qun Zhao, Mengdie Wang, Bowen Zhong, Wenhan Jin, Yanan Huang, Hao Jin, Biao Jing, Wang Wan, Xin Zhang,et al.
American Chemical Society (ACS)
Stress induced amorphous proteome aggregation is a hallmark for diseased cells, with the proteomic composition intimately associated with disease pathogenicity. Due to its particularly dynamic, reversible, and dissociable nature, as well as lack of specific recognition anchor, it is difficult to capture aggregated proteins in situ. In this work, we develop a chemical proteomics method (AggLink) to capture amorphous aggregated proteins in live stressed cells and identify the proteomic contents using LC-MS/MS. Our method relies on an affinity-based chemical probe (AggLink 1.0) that is optimized to selectively bind to and covalently label amorphous aggregated proteins in live stressed cells. Especially, chaotrope-compatible ligation enables effective enrichment of labeled aggregated proteins under urea denaturation and dissociation conditions. Compared to conventional fractionation-based method to profile aggregated proteome, our method showed improved enrichment selectivity, detection sensitivity, and identification accuracy. In HeLa cells, the AggLink method reveals the constituent heterogeneity of aggregated proteome induced by inhibition of pro-folding (HSP90) or pro-degradation (proteasome) pathway, which uncovers a synergistic strategy to reduce cancer cell viability. In addition, the unique fluorogenicity of our probe upon labeling aggregated proteome detects its cellular location and morphology. Together, the AggLink method may help to expand our knowledge of the previously nontargetable amorphous aggregated proteome.
Huan Feng, Qun Zhao, Beirong Zhang, Hang Hu, Meng Liu, Kaifeng Wu, Xiaosong Li, Xin Zhang, Lihua Zhang, and Yu Liu
Wiley
Synthetic fluorescent protein chromophores have been reported for their singlet state fluorescence properties and applications in bioimaging, but rarely for the triplet state chemistries. Herein, we enabled their photo-sensitizing and photo-crosslinking properties through rational modulations. Extension of molecular conjugation and introduction of heavy atoms promoted the generation of reactive oxygen species. Unique from other photosensitizers, these chromophores selectively photo-crosslinked aggregated proteins and uncovered the interactome profiles. We also exemplified their general applications in chromophore-assisted light inactivation, photodynamic therapy and photo induced polymerization. Theoretical calculation, pathway analysis and transient absorption spectra provided mechanistic insights for this triplet state chemistry. Overall, this work expands the function and application of synthetic fluorescent protein chromophores by enabling their triplet excited state properties.
Conner Hoelzel, Yulong Bai, Mengdie Wang, Yu Liu, and Xin Zhang
American Chemical Society (ACS)
Songtao Ye, Andrew P. Latham, Yuqi Tang, Chia-Heng Hsiung, Junlin Chen, Feng Luo, Yu Liu, Bin Zhang, and Xin Zhang
Springer Science and Business Media LLC
Zhiming Zhang, Longchen Zhu, Jiahui Feng, Haoke Zhang, Xin Zhang, Jing Zhi Sun, and Ben Zhong Tang
Royal Society of Chemistry (RSC)
The protein aggregation is successfully monitored using the intrinsic abnormal visible emission at the clustering state, namely clusteroluminescence (CL).
Lihua Liu, Yubo Huang, Yufeng Zhou, Yu Zhao, Jinzhi Qi, Xin Zhang, and Baoxing Shen
Elsevier BV
Eduardo Pinho Melo, Tasuku Konno, Ilaria Farace, Mosab Ali Awadelkareem, Lise R. Skov, Fernando Teodoro, Teresa P. Sancho, Adrienne W. Paton, James C. Paton, Matthew Fares,et al.
Springer Science and Business Media LLC
AbstractProtein synthesis is supported by cellular machineries that ensure polypeptides fold to their native conformation, whilst eliminating misfolded, aggregation prone species. Protein aggregation underlies pathologies including neurodegeneration. Aggregates’ formation is antagonised by molecular chaperones, with cytoplasmic machinery resolving insoluble protein aggregates. However, it is unknown whether an analogous disaggregation system exists in the Endoplasmic Reticulum (ER) where ~30% of the proteome is synthesised. Here we show that the ER of a variety of mammalian cell types, including neurons, is endowed with the capability to resolve protein aggregates under stress. Utilising a purpose-developed protein aggregation probing system with a sub-organellar resolution, we observe steady-state aggregate accumulation in the ER. Pharmacological induction of ER stress does not augment aggregates, but rather stimulate their clearance within hours. We show that this dissagregation activity is catalysed by the stress-responsive ER molecular chaperone – BiP. This work reveals a hitherto unknow, non-redundant strand of the proteostasis-restorative ER stress response.
Lushun Wang, Chia-Heng Hsiung, Xiaojing Liu, Shichao Wang, Axel Loredo, Xin Zhang, and Han Xiao
Royal Society of Chemistry (RSC)
Using a single atom substitution approach, we have developed a series of solvatochromic fluorophores that respond solely to polarity. The utility of these fluorophores is demonstrated by quantifying the polarity of misfolded and aggregated proteins.
Songtao Ye, Chia-Heng Hsiung, Yuqi Tang, and Xin Zhang
American Chemical Society (ACS)
ConspectusProtein aggregation is a biological phenomenon in which aberrantly processed or mutant proteins misfold and assemble into a variety of insoluble aggregates. Decades of studies have delineated the structure, interaction, and activity of proteins in either their natively folded structures or insoluble aggregates such as amyloid fibrils. However, a variety of intermediate species exist between these two extreme states in the protein folding landscape. Herein, we collectively term these intermediate species as misfolded protein oligomers, including soluble oligomers and preamyloid oligomers that are formed by unfolded or misfolded proteins. While extensive tools have been developed to study folded proteins or amyloid fibrils, research to understand the properties and activities of misfolded protein oligomers has been limited by the lack of methods to detect and interrogate these species in live cells.In this Account, we describe our efforts in the development of chemical methods that allow for the characterization of the multistep protein aggregation process, in particular the misfolded protein oligomers, in living cells. As the start of this journey, we attempted to develop a fluorogenic method wherein the misfolded oligomers could turn on the fluorescence of chemical probes that are conjugated to the protein-of-interest (POI). To this end, we produced a series of destabilized HaloTag variants, formulating the primary component of the AgHalo sensor, which misfolds and aggregates when cells are subjected to stress. When AgHalo is covalently conjugated with a solvatochromic fluorophore, misfolding of the AgHalo conjugate would activate fluorescence, resulting in the observation of misfolded oligomers. Following this work, we extended the scope of detection from AgHalo to any protein-of-interest via the AggTag method, wherein the POIs are genetically fused to self-labeling protein tags (HaloTag or SNAP-tag). Focusing on the molecular rotor-based fluorophores, we applied the modulated fluorescent protein (FP) chromophore core as a prototype for the AggTag probes, to enable the fluorogenic detection of misfolded soluble oligomers of multiple proteins in live cells. Next, we further developed the AggTag method to distinguish insoluble aggregates from misfolded oligomers, using two classes of probes that activate different fluorescence emission toward these two conformations. To enable this goal, we applied physical organic chemistry and computational chemistry to discover a new category of triode-like fluorophores, wherein the π orbitals of either an electron density regulator or the donor-acceptor linkages are used to control the rotational barriers of fluorophores in the excited states. This mechanism allows us to rationally design molecular rotor-based fluorophores that have desired responses to viscosity, thus extending the application of the AggTag method.In summary, our work allows the direct monitoring of the misfolded protein oligomers and differentiation of insoluble aggregates from other conformations in live cells, thus enabling studies of many currently unanswered questions in protein aggregation. Future directions are to develop methods that enable quantitative analyses of the protein aggregation process. Further, new methods are needed to detect and to quantify the formation and maturation of protein or RNA condensates that form membraneless organelles.
William Dion, Heather Ballance, Jane Lee, Yinghong Pan, Saad Irfan, Casey Edwards, Michelle Sun, Jing Zhang, Xin Zhang, Silvia Liu,et al.
American Association for the Advancement of Science (AAAS)
A cell-autonomous 12-hour clock coordinates nuclear speckle LLPS with proteostasis control.
Sicheng Tang, Wenting Wang, and Xin Zhang
Elsevier BV
Over the past few years, research tools have been developed to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Herein, we describe recent advances in methods that can either survey the distribution of aggregation at the level of the cellular proteome using mass spectroscopy or discern the multistep aggregation process of specific proteins of interest via fluorescence signals. Future development and application of such technologies are expected to provide insights on mechanisms, diagnosis, and treatment of diseases rooted in protein aggregation.
Yuqian Jiang, Chuanxin Chen, Lauren N. Randolph, Songtao Ye, Xin Zhang, Xiaoping Bao, and Xiaojun Lance Lian
Elsevier BV
Summary Human pluripotent stem cell (hPSC)-derived pancreatic progenitors (PPs) provide promising cell therapies for type 1 diabetes. Current PP differentiation requires a high amount of Activin A during the definitive endoderm (DE) stage, making it economically difficult for commercial ventures. Here we identify a dose-dependent role for Wnt signaling in controlling DE differentiation without Activin A. While high-level Wnt activation induces mesodermal formation, low-level Wnt activation by a small-molecule inhibitor of glycogen synthase kinase 3 is sufficient for DE differentiation, yielding SOX17+FOXA2+ DE cells. BMP inhibition further enhances this DE differentiation, generating over 87% DE cells. These DE cells could be further differentiated into PPs and functional β cells. RNA-sequencing analysis of PP differentiation from hPSCs revealed expected transcriptome dynamics and new gene regulators during our small-molecule PP differentiation protocol. Overall, we established a robust growth-factor-free protocol for generating DE and PP cells, facilitating scalable production of pancreatic cells for regenerative applications.
Sicheng Tang, Songtao Ye, and Xin Zhang
Oxford University Press (OUP)
There is an unmet demand for research tools to monitor the multistep protein aggregation process in live cells, a process that has been associated with a growing number of human diseases. Recently, AIEgens have been developed to directly monitor the entire protein aggregation process in test tubes and live cells. Future application of AIEgens is expected to shed light on both diagnosis and treatment of disease rooted in protein aggregation.