2015 - Present

Cells mitigate ER stress through the unfolded protein response (UPR). Here, we report formation of ER whorls as an effector mechanism of the ER stress response. We found that strong ER stress induces formation of ER whorls, which contain ER-resident proteins such as the Sec61 complex and PKR-like ER kinase (PERK). ER whorl formation is dependent on PERK kinase activity and is mediated by COPII machinery, which facilitates ER membrane budding to form tubular-vesicular ER whorl precursors. ER whorl precursors then go through Sec22b-mediated fusion to form ER whorls. We further show that ER whorls contribute to ER stress-induced translational inhibition by possibly modulating PERK activity and by sequestering translocons in a ribosome-free environment. We propose that formation of ER whorls reflects a new type of ER stress response that controls inhibition of protein translation.

Xu, F., Du, W., Zou, Q., Wang, Y., Zhang, X., Xing, X., Li, Y., Zhang, D., Wang, H., Zhang, W., Hu, X., Liu, X., Liu, X., Zhang, S., Yu, J., Fang, J., Li, F., Zhou, Y., Yue, T., Mi, N., Deng, H., Zou, P., Chen, X., Yang, X.* and Yu, L.* (2020). Cell Res. In press.

DOI: 10.1038/s41422-020-00416-2

The spatial arrangement of chromosome within the nucleus is intimately linked to genome function and gene expression regulation. Existing genome‐wide mapping methods to study chromosome organization often rely on chemically crosslinking DNA with protein baits, which raises concerns of introducing artefacts during cellular fixation. Herein, we developed a novel proximity‐dependent DNA labeling method based on the chromophore‐assisted nucleobase photooxidation. By genetically targeting a photosensitizer protein to specific subnuclear locations, we achieved blue light‐activated labeling of local DNA with a bioorthogonal functional handle, which allowed subsequent affinity purification and sequence identification via next‐generation sequencing. When applied to the nuclear lamina in human embryonic kidney 293T cells, our method revealed lamina‐associated domains (LADs) that cover 37.6% of the genome. These LADs overlap with heterochromatin hallmarks including histone 3 lysine 9 dimethylation (H3K9me2) and are depleted with CpG islands. This simple labeling method avoids the harsh treatment of chemical crosslinking and is generally applicable for the genome‐wide high‐resolution mapping of the spatial chromosome organization in living cells.

Ding, T.#, Zhu, L.#, Fang, Y., Liu, Y., Tang, W. and Zou, P.* (2020). Angew. Chem. Int. Ed. Engl. In press. doi: 10.1002/anie.202005486

DOI: 10.1002/anie.202005486

The subcellular organization of proteins and RNA molecules is crucial for their proper functions. Over the past decade, both ligase-mediated and peroxidase-mediated proximity labeling (PL) techniques have been developed to map biomolecules at near-nanometer spatial resolution and subminute temporal resolution. These methods are shedding light on the spatial arrangement of proteome and transcriptome in their native context. Here, we review the recent evolution and applications of PL techniques, compare and contrast the two classes of methods, and highlight emerging trends and future opportunities.

Zhou, Y. and Zou, P.* (2021). Curr. Opin. Chem. Biol. 60, 30-38.

DOI: 10.1016/j.cbpa.2020.06.013

The engineered ascorbate peroxidase (APEX) is a powerful tool for the proximity-dependent labeling of proteins and RNAs in live cells. Although widely employed in mammalian cells, APEX applications in microorganisms have been hampered by the poor labeling efficiency of its biotin-phenol (BP) substrate. In this study, we sought to address this challenge by designing and screening a panel of alkyne-functionalized substrates. Our best probe, Alk-Ph, substantially improves APEX labeling efficiency in intact yeast cells, as it is more cell wall-permeant than BP. Through a combination of protein-centric and peptide-centric chemoproteomic experiments, we have identified 165 proteins with a specificity of 94% in the yeast mitochondrial matrix. In addition, we have demonstrated that Alk-Ph is useful for proximity-dependent RNA labeling in yeast, thus expanding the scope of APEX-seq. We envision that this improved APEX labeling strategy would set the stage for the large-scale mapping of spatial proteome and transcriptome in yeast.

Li, Y.#, Tian, C., Liu, K., Zhou, Y., Yang, J.* and Zou, P.* (2020). Cell Chem. Biol. 27, 858-865.

DOI: 10.1016/j.chembiol.2020.05.006

Little is known about the underlying mechanisms of the similarities in the core features of postoperative delirium (POD) and α-synuclein (α-syn)-related cognitive disorders. We herein investigated associations between fluctuated levels of exosomal α-syn in the plasma and POD presentation in geriatric hip fracture patients. Exosome α-syn release in plasma may be associated with the POD development which might be due to systemic inflammation.

Yuan, Y.#, Li, Z.#, Yang, N.#, Han, Y., Ji, X., Han, D., Wang, X., Li, Y., Liu, T., Yuan, F., He, J., Liu, Y., Ni, C., Zou, P., Wang, G.*, Guo, X.* and Zhou, Y.* (2020). Front Aging Neurosci. 12, 67.

DOI: 10.3389/fnagi.2020.00067

The occurrence of 8-oxo-7,8-dihydroguanine (OG) in the genome, as one of the major DNA oxidative damages, has been implicated in an array of biological processes, ranging from mutagenesis to transcriptional regulation. Genome-wide mapping of oxidative damages could shed light on the underlying cellular mechanism. In the present study, we engineered the hOGG1 enzyme, a primary 8-oxoguanine DNA glycosylase, into a guanine oxidation-profiling tool. Our method, called enTRAP-seq, successfully identified more than 1,400 guanine oxidation sites in the mouse embryonic fibroblast genome. These OG peaks were enriched in open chromatin regions and regulatory elements including pro-moters, 5’ untranslated regions and CpG islands. Collectively, we present a simple and generalizable approach for the genome-wide profiling of DNA damages with high sensitivity and specificity.

Fang, Y. and Zou, P.* (2020). Biochemistry 59, 85-89.

DOI: 10.1021/acs.biochem.9b00782

Genetically encoded voltage indicators (GEVIs) allow optical recording of neuronal activities with high spatial resolution. While most existing GEVIs emit in the green range, red-shifted GEVIs are highly sought after because they would enable simultaneous stimulation and recording of neuronal activities when paired with optogenetic actuators, or two-color imaging of signaling and neuronal activities when used along with GFP-based indicators. In this study, we present several improved red-shifted GEVIs based on the electrochromic Förster resonance energy transfer (eFRET) between orange/red fluorescent proteins/dyes and rhodopsin mutants. Through structure-guided mutagenesis and cell-based sensitivity screening, we identified a mutant rhodopsin with a single mutation that exhibited more than 2-fold improvement in voltage sensitivity. Notably, this mutation has been independently discovered by Pieribone et al. (Pieribone, V. A. et al. Nat Methods 2018, 15 (12), 1108−1116). In cultured rat hippocampal neurons, our sensors faithfully reported action potential waveforms and subthreshold activities. We also demonstrated that this mutation could enhance the sensitivity of hybrid indicators, thus providing insights for future development.

Xu, Y.#, Deng, M.#, Zhang, S.#, Yang, J.#, Peng, L., Chu, J.* and Zou, P.* (2019). ACS Chem. Neurosci. 10, 4768-4775.

DOI: 10.1021/acschemneuro.9b00501

Biological macromolecules (proteins, nucleic acids, polysaccharides, etc.) are the building blocks of life, which constantly undergo chemical modifications that are often reversible and spatial-temporally regulated. These dynamic properties of chemical modifications play fundamental roles in physiological processes as well as pathological changes of living systems. The Major Research Project (MRP) funded by the National Natural Science Foundation of China (NSFC)-"Dynamic modifications of biomacromolecules: mechanism and chemical interventions" aims to integrate cross-disciplinary approaches at the interface of chemistry, life sciences, medicine, mathematics, material science and information science with the following goals: (i) developing specific labeling techniques and detection methods for dynamic chemical modifications of biomacromolecules, (ii) analyzing the molecular mechanisms and functional relationships of dynamic chemical modifications of biomacromolecules, and (iii) exploring biomacromolecules and small molecule probes as potential drug targets and lead compounds.

Wang, C., Zou, P., Yang, C., Liu, L., Cheng, L., He, X., Zhang, L., Zhang, Y., Jiang, H.* and Chen, P.R.* (2019). Sci. China Life Sci. 62, 1459-1471.

DOI: 10.1007/s11427-019-9823-1

RNA molecules are highly compartmentalized in eukaryotic cells, with their localizations intimately linked to their functions. Despite the importance of RNA targeting, our current knowledge of the spatial organization of transcriptome has been limited by a lack of analytical tools. In this study, we develop a novel chemical biology approach to label RNAs in live cells with high spatial specificity. Our method, called CAP-seq, capitalizes on light-activated, proximity-dependent photo-oxidation of RNA nucleobases, which could be subsequently enriched via affinity purification and identified by high-throughput sequencing. Using this technique, we investigate the local transcriptomes that are proximal to various subcellular compartments, including the endoplasmic reticulum and mitochondria. We discover that mRNAs encoding for ribosomal proteins and oxidative phosphorylation pathway proteins are highly enriched at the outer mitochondrial membrane. Due to its specificity and ease of use, CAP-seq is a generally applicable technique to investigate the spatial transcriptome in many biological systems.

Wang, P.#, Tang, W.#, Li, Z.#, Zou, Z., Zhou, Y., Li, R., Xiong, T., Wang, J.* and Zou, P.* (2019). Nat. Chem. Biol. 15, 1110-1119.

DOI: 10.1038/s41589-019-0368-5

Patients with liver diseases often suffer from chronic itch, yet the pruritogen(s) and receptor(s) remain largely elusive. Here, we identify bile acids as natural ligands for MRGPRX4. MRGPRX4 is expressed in human dorsal root ganglion (hDRG) neurons and co-expresses with itch receptor HRH1. Bile acids elicited Ca2+ responses in cultured hDRG neurons, and bile acids or a MRGPRX4 specific agonist induced itch in human subjects. However, a specific agonist for another bile acid receptor TGR5 failed to induce itch in human subjects and we find that human TGR5 is not expressed in hDRG neurons. Finally, we show positive correlation between cholestatic itch and plasma bile acids level in itchy patients and the elevated bile acids is sufficient to activate MRGPRX4. Taken together, our data strongly suggest that MRGPRX4 is a novel bile acid receptor that likely underlies cholestatic itch in human, providing a promising new drug target for anti-itch therapies.

Yu, H., Zhao, T., Liu, S., Wu, Q., Johnson, O., Wu, Z., Zhuang, Z., Shi, Y., Peng, L., He, R., Yang, Y., Sun, J., Wang, X., Xu, H., Zeng, Z., Zou, P., Lei, X., Luo, W.* and Li, Y.* (2019). Elife 8, e48431.

DOI: 10.7554/eLife.48431

The subcellular organization of biomolecules such as proteins and nucleic acids is intimately linked to their biological functions. APEX2, an engineered ascorbate peroxidase that enables proximity-dependent labeling of proteins in living cells, has emerged as a powerful tool for deciphering the molecular architecture of various subcellular structures. However, only phenolic compounds have thus far been employed as APEX2 substrates, and the resulting phenoxyl radicals preferentially react with electron-rich amino acid residues. This narrow scope of substrates could potentially limit the application of APEX2. In this study, we screened a panel of aromatic compounds and identified biotin-conjugated arylamines as novel probes with significantly higher reactivity towards nucleic acids. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we applied APEX2 labeling with biotin-aniline (Btn-An) in the mitochondrial matrix, capturing all 13 mitochondrial messenger RNAs and none of the cytoplasmic RNAs. APEX2-mediated Btn-An labeling of RNA is thus a promising method for mapping the subcellular transcriptome, which could shed light on their functions in cell physiology.

Zhou, Y.#, Wang, G.#, Wang, P., Li, Z., Yue, T., Wang, J. and Zou, P.* (2019). Angew. Chem. Int. Ed. Engl. 58, 11763-11767.

DOI: 10.1002/anie.201905949

In living organisms, protein functions are constantly evolving over generations throughout the history. Through iterative rounds of genetic mutations and natural selection of fit phenotypes, protein functions have been gradually optimized. This process could be mimicked and even greatly accelerated in the laboratory, when the selection pressure is directly applied to biomolecules of interest, which forms the basis of a technique called directed evolution. The Nobel Prize in chemistry 2018 was awarded jointly to Frances Arnold, George Smith and Gregory P. Winter for their pioneering contributions to the development and applications of directed evolution. Here we briefly review the history of this technique and its impact on renewable energy and pharmaceutical industry.

Zhou, Y.#, Zhu, L.# and Zou, P.* (2019). Univ. Chem. 34, 1-6.

DOI: 10.3866/PKU.DXHX201811008

Human induced pluripotent stem cell (iPSC)-derived neurons are an attractive substrate for modeling disease, yet the heterogeneity of these cultures presents a challenge for functional characterization by manual patch-clamp electrophysiology. Here, we describe an optimized all-optical electrophysiology, "Optopatch," pipeline for high-throughput functional characterization of human iPSC-derived neuronal cultures. We demonstrate the method in a human iPSC-derived motor neuron (iPSC-MN) model of amyotrophic lateral sclerosis (ALS). In a comparison of iPSC-MNs with an ALS-causing mutation (SOD1 A4V) with their genome-corrected controls, the mutants showed elevated spike rates under weak or no stimulus and greater likelihood of entering depolarization block under strong optogenetic stimulus. We compared these results with numerical simulations of simple conductance-based neuronal models and with literature results in this and other iPSC-based models of ALS. Our data and simulations suggest that deficits in slowly activating potassium channels may underlie the changes in electrophysiology in the SOD1 A4V mutation.

Kiskinis, E.#, Kralj, J. M.#, Zou, P.#, Weinstein, E. N.#, Zhang, H., Tisoras, K., Wiskow, O., Ortega, J. A., Eggan, K.* and Cohen, A. E.* (2018). Stem Cell Reports 10, 1991-2004.

DOI: 10.1016/j.stemcr.2018.04.020

Optical biosensors have been invaluable tools in neuroscience research, as they provide the ability to directly visualize neural activity in real time, with high specificity, and with exceptional spatial and temporal resolution. Notably, a majority of these sensors are based on fluorescent protein scaffolds, which offer the ability to target specific cell types or even subcellular compartments. However, fluorescent proteins are intrinsically bulky tags, often insensitive to the environment, and always require excitation light illumination. To address these limitations, there has been a proliferation of alternative sensor scaffolds developed in recent years, including hybrid sensors that combine the advantages of synthetic fluorophores and genetically encoded protein tags, as well as bioluminescent probes. While still in their early stage of development as compared with fluorescent protein-based sensors, these novel probes have offered complementary solutions to interrogate various aspects of neuronal communication, including transmitter release, changes in membrane potential, and the production of second messengers. In this Review, we discuss these important new developments with a particular focus on design strategies.

Wang, A.#, Feng, J.#, Li, Y.* and Zou, P.* (2018). ACS. Chem. Neurosci. 9, 639-650.

DOI: 10.1021/acschemneuro.7b00455

Membrane voltage is an important biophysical signal that underlies intercellular electrical communications. In this study, we present a novel fluorescent voltage indicator that enables the investigation of electrical signaling at high spatial resolution. Our method is built upon the site-specific modification of microbial rhodopsin proteins with organic fluorophores, resulting in a hybrid indicator scaffold that represents one of the most sensitive and fastest orange-colored voltage indicators developed to date. We applied this technique to optically map electrical connectivity in cultured cells, which revealed gap junction-mediated long-range coupling that spanned over hundreds of micrometers.

Xu, Y.#, Peng, L.#, Wang, S.#, Wang, A.#, Ma, R., Zhou, Y., Yang, J., Sun, D. E., Lin, W., Chen, X. and Zou, P.* (2018). Angew. Chem. Int. Ed. Engl. 57, 3949-3553.

DOI: 10.1002/anie.201712614

We developed membrane voltage nanosensors that are based on inorganic semiconductor nanoparticles. We provide here a feasibility study for their utilization. We use a rationally designed peptide to functionalize the nanosensors, imparting them with the ability to self-insert into a lipid membrane with a desired orientation. Once inserted, these nanosensors could sense membrane potential via the quantum confined Stark effect, with a single-particle sensitivity. With further improvements, these nanosensors could potentially be used for simultaneous recording of action potentials from multiple neurons in a large field of view over a long duration and for recording electrical signals on the nanoscale, such as across one synapse.

Park, K., Kuo, Y., Shvadchak, V., Ingargiola, A., Dai, X., Hsiung, L., Kim, W., Zhou, H., Zou, P., Levine, A. J., Li, J. and Weiss, S.* (2018). Sci. Adv. 4, e1601453.

DOI: 10.1126/sciadv.1601453

A holy grail in neuroscience is to understand how brain functions arise from neural network-level electrical activities. Voltage imaging allows for the direct visualization of electrical signaling at high spatial and temporal resolutions across a large neuronal population. Central to this technique is a palette of genetically-encoded fluorescent probes with fast and sensitive voltage responses. In this review, we chronicle the development and applications of genetically-encoded voltage indicators (GEVIs) over the past two decades, with a primary focus on the structural design that harness the power of fluctuating transmembrane electric fields. We hope this article will inform chemical biologists and protein engineers of the GEVI history and inspire novel design ideas.

Peng, L.#, Xu, Y.# and Zou, P.* (2017). Chin. Chem. Lett. 28, 1925-1928.

DOI: 10.1016/j.cclet.2017.09.037

Membrane voltages are ubiquitous throughout cell biology. Voltage is most commonly associated with excitable cells such as neurons and cardiomyocytes, although many other cell types and organelles also support electrical signaling. Voltage imaging in vivo would offer unique capabilities in reporting the spatial pattern and temporal dynamics of electrical signaling at the cellular and circuit levels. Voltage is not directly visible, and so a longstanding challenge has been to develop genetically encoded fluorescent voltage indicator proteins. Recent advances have led to a profusion of new voltage indicators, based on different scaffolds and with different tradeoffs between voltage sensitivity, speed, brightness, and spectrum. In this review, we describe recent advances in design and applications of genetically-encoded voltage indicators (GEVIs). We also highlight the protein engineering strategies employed to improve the dynamic range and kinetics of GEVIs and opportunities for future advances.

Xu, Y., Zou, P.* and Cohen, A. E.* (2017). Curr. Opin. Chem. Biol. 39, 1-10.

DOI: 10.1016/j.cbpa.2017.04.005

Optical imaging of voltage indicators based on green fluorescent proteins (FPs) or archaerhodopsin has emerged as a powerful approach for detecting the activity of many individual neurons with high spatial and temporal resolution. Relative to green FP-based voltage indicators, a bright red-shifted FP-based voltage indicator has the intrinsic advantages of lower phototoxicity, lower autofluorescent background, and compatibility with blue-light-excitable channelrhodopsins. Here, we report a bright red fluorescent voltage indicator (fluorescent indicator for voltage imaging red; FlicR1) with properties that are comparable to the best available green indicators. To develop FlicR1, we used directed protein evolution and rational engineering to screen libraries of thousands of variants. FlicR1 faithfully reports single action potentials (~3% ΔF/F) and tracks electrically driven voltage oscillations at 100 Hz in dissociated Sprague Dawley rat hippocampal neurons in single trial recordings. Furthermore, FlicR1 can be easily imaged with wide-field fluorescence microscopy. We demonstrate that FlicR1 can be used in conjunction with a blue-shifted channelrhodopsin for all-optical electrophysiology, although blue light photoactivation of the FlicR1 chromophore presents a challenge for applications that require spatially overlapping yellow and blue excitation.

Abdelfattah, A. S., Farhi, S. L., Zhao, Y., Brinks, D., Zou, P., Ruangkittisakul, A., Platisa, J., Pieribone, V. A., Ballanyi, K., Cohen, A. E. and Campbell, R. E.* (2016). J Neurosci. 36, 2458-2472.

DOI: 10.1523/JNEUROSCI.3484-15.2016

Before 2015

All-optical electrophysiology—spatially resolved simultaneous optical perturbation and measurement of membrane voltage—would open new vistas in neuroscience research. We evolved two archaerhodopsin-based voltage indicators, QuasAr1 and QuasAr2, which show improved brightness and voltage sensitivity, have microsecond response times and produce no photocurrent. We engineered a channelrhodopsin actuator, CheRiff, which shows high light sensitivity and rapid kinetics and is spectrally orthogonal to the QuasArs. A coexpression vector, Optopatch, enabled cross-talk–free genetically targeted all-optical electrophysiology. In cultured rat neurons, we combined Optopatch with patterned optical excitation to probe back-propagating action potentials (APs) in dendritic spines, synaptic transmission, subcellular microsecond-timescale details of AP propagation, and simultaneous firing of many neurons in a network. Optopatch measurements revealed homeostatic tuning of intrinsic excitability in human stem cell–derived neurons. In rat brain slices, Optopatch induced and reported APs and subthreshold events with high signal-to-noise ratios. The Optopatch platform enables high-throughput, spatially resolved electrophysiology without the use of conventional electrodes.

Hochbaum, D. R.#, Zhao, Y.#, Farhi, S. L., Klapoetke, N., Werley, C. A., Kapoor, V., Zou, P., Kralj, J. M., Maclaurin, D., Smedemark-Margulies, N., Saulnier, J. L., Boulting, G. L., Straub, C., Cho, Y. K., Melkonian, M., Wong, G. K. S., Harrison, D. J., Murthy, V. N., Sabatini, B. L., Boyden, E. S., Campbell, R. E. and Cohen, A. E.* (2014). Nat. Methods 11, 825-833.

DOI: 10.1038/nmeth.3000

Genetically encoded fluorescent reporters of membrane potential promise to reveal aspects of neural function not detectable by other means. We present a palette of multicoloured brightly fluorescent genetically encoded voltage indicators with sensitivities from 8–13% ΔF/F per 100 mV, and half-maximal response times from 4–7 ms. A fluorescent protein is fused to an archaerhodopsin-derived voltage sensor. Voltage-induced shifts in the absorption spectrum of the rhodopsin lead to voltage-dependent nonradiative quenching of the appended fluorescent protein. Through a library screen, we identify linkers and fluorescent protein combinations that report neuronal action potentials in cultured rat hippocampal neurons with a single-trial signal-to-noise ratio from 7 to 9 in a 1 kHz imaging bandwidth at modest illumination intensity. The freedom to choose a voltage indicator from an array of colours facilitates multicolour voltage imaging, as well as combination with other optical reporters and optogenetic actuators.

Zou, P.#, Zhao, Y. #, Douglass, A. D., Hochbaum, D. R., Brinks, D., Werley, C. A., Harrison, D. J., Campbell, R. E.* and Cohen, A. E.* (2014). Nat. Commun. 5, 4625.

DOI: 10.1038/ncomms5625

Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.

Hung, V., Zou, P., Rhee, H., Udeshi, N. D., Cracan, V., Svinkina, T., Carr, S. A., Mootha, V. K. and Ting, A. Y.* (2014). Mol. Cell 55, 332-341.

DOI: 10.1016/j.molcel.2014.06.003

The low-density lipoprotein receptor (LDLR) is a critical determinant of plasma cholesterol levels that internalizes lipoprotein cargo via clathrin-mediated endocytosis. Here, we show that the E3 ubiquitin ligase IDOL stimulates a previously unrecognized, clathrin-independent pathway for LDLR internalization. Real-time single-particle tracking and electron microscopy reveal that IDOL is recruited to the plasma membrane by LDLR, promotes LDLR internalization in the absence of clathrin or caveolae, and facilitates LDLR degradation by shuttling it into the multivesicular body (MVB) protein-sorting pathway. The IDOL-dependent degradation pathway is distinct from that mediated by PCSK9 as only IDOL employs ESCRT (endosomal-sorting complex required for transport) complexes to recognize and traffic LDLR to lysosomes. Small interfering RNA (siRNA)-mediated knockdown of ESCRT-0 (HGS) or ESCRT-I (TSG101) components prevents IDOL-mediated LDLR degradation. We further show that USP8 acts downstream of IDOL to deubiquitinate LDLR and that USP8 is required for LDLR entry into the MVB pathway. These results provide key mechanistic insights into an evolutionarily conserved pathway for the control of lipoprotein receptor expression and cellular lipid uptake.

Scotti, E., Calamai, M., Goulbourne, C. N., Zhang, L., Hong, C., Lin, R. R., Choi, J., Pilch, P. F., Fong, L. G., Zou, P., Ting, A. Y., Pavone, F. S., Young, S. G. and Tontonoz, P.* (2013). Mol. Cell. Biol. 33, 1503-1514.

DOI: 10.1128/MCB.01716-12

Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

Rhee, H. W.#, Zou, P.#, Udeshi, N. D., Martell, J. D., Mootha, V. K., Carr, S. A. and Ting, A. Y.* (2013). Science 339, 1328-1331.

DOI: 10.1126/science.1230593

The C-terminal domain (Mpro-C) of SARS-CoV main protease adopts two different fold topologies, a monomer and a 3D domain-swapped dimer. Here, we report that Mpro-C can reversibly interconvert between these two topological states under physiological conditions. Although the swapped α1-helix is fully buried inside the protein hydrophobic core, the interconversion of Mpro-C is carried out without the hydrophobic core being exposed to solvent. The 3D domain swapping of Mpro-C is activated by an order-to-disorder transition of its C-terminal α5-helix foldon. Unfolding of this foldon promotes self-association of Mpro-C monomers and functions to mediate the 3D domain swapping, without which Mpro-C can no longer form the domain-swapped dimer. Taken together, we propose that there exists a special dimeric intermediate enabling the protein core to unpack and the α1-helices to swap in a hydrophobic environment, which minimizes the energy cost of the 3D domain-swapping process.

Kang, X., Zhong, N., Zou, P., Zhang, S. N., Jin, C. W. and Xia, B.* (2012). Proc. Natl. Acad. Sci. U. S. A. 109, 14900-14905.

DOI: 10.1073/pnas.1205241109

A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for AlexaFluor568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, we labeled the low-density lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-molecule imaging and tracking over time.

Cohen, J. D., Zou, P. and Ting, A. Y.* (2012). Chembiochem 13, 888-894.

DOI: 10.1002/cbic.201100764

Methods to probe receptor oligomerization are useful to understand the molecular mechanisms of receptor signaling. Here we report a fluorescence imaging method to determine receptor oligomerization state in living cells during endocytic internalization. The wild-type receptor is co-expressed with an internalization-defective mutant, and the internalization kinetics of each are independently monitored. If the receptor internalizes as an oligomer, then the wild-type and mutant isoforms will mutually influence each others’ trafficking properties, causing co-internalization of the mutant or co-retention of the wild-type at the cell surface. Using this approach, we found that the low density lipoprotein (LDL) receptor internalizes as an oligomer into cells, both in the presence and absence of LDL ligand. The internalization kinetics of the wild-type receptor are not changed by LDL binding. We also found that the oligomerization domain of the LDL receptor is located in its cytoplasmic tail.

Zou, P. and Ting, A. Y.* (2011). ACS Chem. Biol. 6, 308-313.

DOI: 10.1021/cb100361k

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGF-like domain of HB-EGF and the cytoplasmic C-terminus. A striking observation was the absence of the HB-EGF transmembrane pro-form from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HB-EGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.

Prince, R. N., Schreiter, E. R., Zou, P., Wiley, H. S., Ting, A. Y., Lee, R. T. and Lauffenburger, D. A.* (2010). J. Cell. Sci. 123, 2308-2318.

DOI: 10.1242/jcs.058321

SARS coronavirus main protease (Mpro) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. We have reported that both the Mpro C-terminal domain alone (Mpro-C) and the N-finger deletion mutant of Mpro (Mpro-Δ7) exist as a stable dimer and a stable monomer (Zhong et al., J Virol 2008; 82:4227-4234). Here, we report structures of both Mpro-C monomer and dimer. The structure of the Mpro-C monomer is almost identical to that of the C-terminal domain in the crystal structure of Mpro. Interestingly, the Mpro-C dimer structure is characterized by 3D domain-swapping, in which the first helices of the two protomers are interchanged and each is enwrapped by four other helices from the other protomer. Each folding subunit of the Mpro-C domain-swapped dimer still has the same general fold as that of the Mpro-C monomer. This special dimerization elucidates the structural basis for the observation that there is no exchange between monomeric and dimeric forms of Mpro-C and Mpro-Δ7.

Zhong, N.#, Zhang, S. N.#, Xue, F.#, Kang, X., Zou, P., Chen, J. X., Liang, C., Rao, Z. H., Jin, C. W., Lou, Z. Y. and Xia, B. (2009). Protein Sci. 18, 839-844.

DOI: 10.1002/pro.76

The main protease (Mpro) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays an essential role in the extensive proteolytic processing of the viral polyproteins (pp1a and pp1ab), and it is an important target for anti-SARS drug development. It was found that SARS-CoV Mpro exists in solution as an equilibrium of both monomeric and dimeric forms, and the dimeric form is the enzymatically active form. However, the mechanism of SARS-CoV Mpro dimerization, especially the roles of its N-terminal seven residues (N-finger) and its unique C-terminal domain in the dimerization, remain unclear. Here we report that the SARS-CoV Mpro C-terminal domain alone (residues 187 to 306; Mpro-C) is produced in Escherichia coli in both monomeric and dimeric forms, and no exchange could be observed between them at room temperature. The Mpro-C dimer has a novel dimerization interface. Meanwhile, the N-finger deletion mutant of SARS-CoV Mpro also exists as both a stable monomer and a stable dimer, and the dimer is formed through the same C-terminal-domain interaction as that in the Mpro-C dimer. However, no C-terminal domain-mediated dimerization form can be detected for wild-type SARS-CoV Mpro. Our study results help to clarify previously published controversial claims about the role of the N-finger in SARS-CoV Mpro dimerization. Apparently, without the N-finger, SARS-CoV Mpro can no longer retain the active dimer structure; instead, it can form a new type of dimer which is inactive. Therefore, the N-finger of SARS-CoV Mpro is not only critical for its dimerization but also essential for the enzyme to form the enzymatically active dimer.

Zhong, N., Zhang, S. N., Zou, P., Chen, J. X., Kang, X., Li, Z., Liang, C., Jin, C. W. and Xia, B.* (2008). J. Virol. 82, 4227-4234.

DOI: 10.1128/JVI.02612-07