Doctor of Philosophy, University of California Los Angeles (2011)
Bachelor of Science, Fudan University (2005)
Axel Brunger, Postdoctoral Faculty Sponsor
In synaptic terminals, complexin is thought to have inhibitory and activating roles for spontaneous "mini" release and evoked synchronized neurotransmitter release, respectively. We used single vesicle-vesicle microscopy imaging to study the effect of complexin-1 on the on-rate of docking between vesicles that mimic synaptic vesicles and the plasma membrane. We found that complexin-1 enhances the on-rate of docking of synaptic vesicle mimics containing full-length synaptobrevin-2 and full-length synaptotagmin-1 to plasma membrane-mimicking vesicles containing full-length syntaxin-1A and SNAP-25A. This effect requires the C-terminal domain of complexin-1, which binds to the membrane, the presence of PS in the membrane, and the core region of complexin-1, which binds to the SNARE complex.
View details for DOI 10.1021/ja407392n
View details for PubMedID 24083833
In vitro reconstitution assays are commonly used to study biological membrane fusion. However, to date, most ensemble and single-vesicle experiments involving SNARE proteins have been performed only with lipid-mixing, but not content-mixing indicators. Through simultaneous detection of lipid and small content-mixing indicators, we found that lipid mixing often occurs seconds prior to content mixing, or without any content mixing at all, during a 50-seconds observation period, for Ca(2+) -triggered fusion with SNAREs, full-length synaptotagmin-1, and complexin. Our results illustrate the caveats of commonly used bulk lipid-mixing fusion experiments. We recommend that proteoliposome fusion experiments should always employ content-mixing indicators in addition to, or in place of, lipid-mixing indicators.
View details for DOI 10.1002/bies.201300010
View details for Web of Science ID 000320394000012
View details for PubMedID 23625805
Amyloid protein aggregates are associated with dozens of devastating diseases including Alzheimer's, Parkinson's, ALS, and diabetes type 2. While structure-based discovery of compounds has been effective in combating numerous infectious and metabolic diseases, ignorance of amyloid structure has hindered similar approaches to amyloid disease. Here we show that knowledge of the atomic structure of one of the adhesive, steric-zipper segments of the amyloid-beta (Aβ) protein of Alzheimer's disease, when coupled with computational methods, identifies eight diverse but mainly flat compounds and three compound derivatives that reduce Aβ cytotoxicity against mammalian cells by up to 90%. Although these compounds bind to Aβ fibers, they do not reduce fiber formation of Aβ. Structure-activity relationship studies of the fiber-binding compounds and their derivatives suggest that compound binding increases fiber stability and decreases fiber toxicity, perhaps by shifting the equilibrium of Aβ from oligomers to fibers. DOI:http://dx.doi.org/10.7554/eLife.00857.001.
View details for DOI 10.7554/eLife.00857
View details for PubMedID 23878726
Although aberrant protein aggregation has been conclusively linked to dozens of devastating amyloid diseases, scientists remain puzzled about the molecular features that render amyloid fibrils or small oligomers toxic. Here, we report a previously unobserved type of amyloid fibril that tests as cytotoxic: one in which the strands of the contributing ?-sheets are out of register. In all amyloid fibrils previously characterized at the molecular level, only in-register ?-sheets have been observed, in which each strand makes its full complement of hydrogen bonds with the strands above and below it in the fibril. In out-of-register sheets, strands are sheared relative to one another, leaving dangling hydrogen bonds. Based on this finding, we designed out-of-register ?-sheet amyloid mimics, which form both cylindrin-like oligomers and fibrils, and these mimics are cytotoxic. Structural and energetic considerations suggest that out-of-register fibrils can readily convert to toxic cylindrins. We propose that out-of-register ?-sheets and their related cylindrins are part of a toxic amyloid pathway, which is distinct from the more energetically favored in-register amyloid pathway.
View details for DOI 10.1073/pnas.1218792109
View details for Web of Science ID 000313123700039
View details for PubMedID 23213214
The amyloid protein aggregation associated with diseases such as Alzheimer's, Parkinson's and type II diabetes (among many others) features a bewildering variety of ?-sheet-rich structures in transition from native proteins to ordered oligomers and fibres. The variation in the amino-acid sequences of the ?-structures presents a challenge to developing a model system of ?-sheets for the study of various amyloid aggregates. Here, we introduce a family of robust ?-sheet macrocycles that can serve as a platform to display a variety of heptapeptide sequences from different amyloid proteins. We have tailored these amyloid ?-sheet mimics (ABSMs) to antagonize the aggregation of various amyloid proteins, thereby reducing the toxicity of amyloid aggregates. We describe the structures and inhibitory properties of ABSMs containing amyloidogenic peptides from the amyloid-? peptide associated with Alzheimer's disease, ?(2)-microglobulin associated with dialysis-related amyloidosis, ?-synuclein associated with Parkinson's disease, islet amyloid polypeptide associated with type II diabetes, human and yeast prion proteins, and Tau, which forms neurofibrillary tangles.
View details for DOI 10.1038/NCHEM.1433
View details for Web of Science ID 000310436600012
View details for PubMedID 23089868
Amyloid diseases, including Alzheimer's, Parkinson's, and the prion conditions, are each associated with a particular protein in fibrillar form. These amyloid fibrils were long suspected to be the disease agents, but evidence suggests that smaller, often transient and polymorphic oligomers are the toxic entities. Here, we identify a segment of the amyloid-forming protein ?B crystallin, which forms an oligomeric complex exhibiting properties of other amyloid oligomers: ?-sheet-rich structure, cytotoxicity, and recognition by an oligomer-specific antibody. The x-ray-derived atomic structure of the oligomer reveals a cylindrical barrel, formed from six antiparallel protein strands, that we term a cylindrin. The cylindrin structure is compatible with a sequence segment from the ?-amyloid protein of Alzheimer's disease. Cylindrins offer models for the hitherto elusive structures of amyloid oligomers.
View details for DOI 10.1126/science.1213151
View details for Web of Science ID 000301225100046
View details for PubMedID 22403391
Type IV pili (TFP) and exopolysaccharides (EPS) are important components for social behaviors in Myxococcus xanthus, including gliding motility and fruiting body formation. Although specific interactions between TFP and EPS have been proposed, there have as yet been no direct observations of these interactions under native conditions. In this study, we found that a truncated PilA protein (PilACt) containing only the C-terminal domain (amino acids 32-208) is sufficient for EPS binding in vitro. Furthermore, an enhanced green fluorescent protein (eGFP) and PilACt fusion protein were constructed and used to label the native EPS in M. xanthus. Under confocal laser scanning microscope, the eGFP-PilACt-bound fruiting bodies, trail structures and biofilms exhibited similar patterns as the wheat germ agglutinin lectin-labeled EPS structures. This study showed that eGFP-PilACt fusion protein was able efficiently to label the EPS of M. xanthus, providing evidence for the first time of the direct interaction between the PilA protein and EPS under native conditions.
View details for DOI 10.1111/j.1574-6968.2011.02430.x
View details for Web of Science ID 000297585000004
View details for PubMedID 22092602
Combining the concepts of synthetic symmetrization with the approach of engineering metal-binding sites, we have developed a new crystallization methodology termed metal-mediated synthetic symmetrization. In this method, pairs of histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metal. Metal-mediated synthetic symmetrization greatly expands the packing and oligomeric assembly possibilities of target proteins, thereby increasing the chances of growing diffraction-quality crystals. To demonstrate this method, we designed various T4 lysozyme (T4L) and maltose-binding protein (MBP) mutants and cocrystallized them with one of three metal ions: copper (Cu²?, nickel (Ni²?), or zinc (Zn²?). The approach resulted in 16 new crystal structures--eight for T4L and eight for MBP--displaying a variety of oligomeric assemblies and packing modes, representing in total 13 new and distinct crystal forms for these proteins. We discuss the potential utility of the method for crystallizing target proteins of unknown structure by engineering in pairs of histidine or cysteine residues. As an alternate strategy, we propose that the varied crystallization-prone forms of T4L or MBP engineered in this work could be used as crystallization chaperones, by fusing them genetically to target proteins of interest.
View details for DOI 10.1002/pro.727
View details for Web of Science ID 000296273700014
View details for PubMedID 21898649
Amyloid-beta (A?) aggregates are the main constituent of senile plaques, the histological hallmark of Alzheimer's disease. A? molecules form ?-sheet containing structures that assemble into a variety of polymorphic oligomers, protofibers, and fibers that exhibit a range of lifetimes and cellular toxicities. This polymorphic nature of A? has frustrated its biophysical characterization, its structural determination, and our understanding of its pathological mechanism. To elucidate A? polymorphism in atomic detail, we determined eight new microcrystal structures of fiber-forming segments of A?. These structures, all of short, self-complementing pairs of ?-sheets termed steric zippers, reveal a variety of modes of self-association of A?. Combining these atomic structures with previous NMR studies allows us to propose several fiber models, offering molecular models for some of the repertoire of polydisperse structures accessible to A?. These structures and molecular models contribute fundamental information for understanding A? polymorphic nature and pathogenesis.
View details for DOI 10.1073/pnas.1112600108
View details for Web of Science ID 000295973800020
View details for PubMedID 21949245
Aggregates of the protein ?-synuclein are the main component of Lewy bodies, the hallmark of Parkinson's disease. ?-Synuclein aggregates are also found in many human neurodegenerative diseases known as synucleinopathies. In vivo, ?-synuclein associates with membranes and adopts ?-helical conformations. The details of how ?-synuclein converts from the functional native state to amyloid aggregates remain unknown. In this study, we use maltose-binding protein (MBP) as a carrier to crystallize segments of ?-synuclein. From crystal structures of fusions between MBP and four segments of ?-synuclein, we have been able to trace a virtual model of the first 72 residues of ?-synuclein. Instead of a mostly ?-helical conformation observed in the lipid environment, our crystal structures show ?-helices only at residues 1-13 and 20-34. The remaining segments are extended loops or coils. All of the predicted fiber-forming segments based on the 3D profile method are in extended conformations. We further show that the MBP fusion proteins with fiber-forming segments from ?-synuclein can also form fiber-like nano-crystals or amyloid-like fibrils. Our structures suggest intermediate states during amyloid formation of ?-synuclein.
View details for DOI 10.1002/pro.630
View details for Web of Science ID 000291068000006
View details for PubMedID 21462277
Gammaherpesviruses Kaposi's sarcoma-associated herpesvirus and Epstein-Barr virus are associated with multiple human cancers. Our goal was to develop a quantitative, high-throughput functional profiling system to identify viral cis-elements and protein subdomains critical for virus replication in the context of the herpesvirus genome. In gamma-2 herpesviruses, the transactivating factor RTA is essential for initiation of lytic gene expression and viral reactivation. We used the RTA locus as a model to develop the functional profiling approach. The mutant murine gammaherpesvirus 68 viral library, containing 15-bp random insertions in the RTA locus, was passaged in murine fibroblast cells for multiple rounds of selection. The effect of each 15-bp insertion was characterized using fluorescent-PCR profiling. We identified 1,229 insertions in the 3,845-bp RTA locus, of which 393, 282, and 554 were critically impaired, attenuated, and tolerated, respectively, for viral growth. The functional profiling phenotypes were verified by examining several individual RTA mutant clones for transactivating function of the RTA promoter and transcomplementing function of the RTA-null virus. Thus, the profiling approach enabled us to identify several novel functional domains in the RTA locus in the context of the herpesvirus genome. Importantly, our study has demonstrated a novel system to conduct high-density functional genetic mapping. The genome-scale expansion of the genetic profiling approach will expedite the functional genomics research on herpesvirus.
View details for DOI 10.1128/JVI.02302-08
View details for Web of Science ID 000262840200027
View details for PubMedID 19073723
A baculoviral mammalian-cell vector was constructed to express Rta, a protein of Epstein-Barr virus (EBV) responsible for the transition from latent infection to lytic replication. EBV lytic replication and cell-growth inhibition was observed in infected D98/HR1 cells. The baculovirus caused little cytotoxicity in the non-targeted HeLa cells, compared to an adenovirus vector. It is concluded that recombinant baculovirus might have the potential as a vector for the therapy of EBV-related cancer.
View details for DOI 10.1007/s00705-006-0772-5
View details for Web of Science ID 000240723300012
View details for PubMedID 16673043