All Publications

  • Determining composition of micron-scale protein deposits in neurodegenerative disease by spatially targeted optical microproteomics. eLife Hadley, K. C., Rakhit, R., Guo, H., Sun, Y., Jonkman, J. E., McLaurin, J., Hazrati, L. N., Emili, A., Chakrabartty, A. 2015; 4


    Spatially targeted optical microproteomics (STOMP) is a novel proteomics technique for interrogating micron-scale regions of interest (ROI) in mammalian tissue, with no requirement for genetic manipulation. Methanol or formalin fixed specimens are stained with fluorescent dyes or antibodies to visualize ROIs, then soaked in solutions containing the photo-tag: 4-benzoylbenzyl-glycyl-hexahistidine. Confocal imaging along with two photon excitation are used to covalently couple photo-tags to all proteins within each ROI, to a resolution of 0.67 µm in the xy-plane and 1.48 µm axially. After tissue solubilization, photo-tagged proteins are isolated and identified by mass spectrometry. As a test case, we examined amyloid plaques in an Alzheimer's disease (AD) mouse model and a postmortem AD case, confirming known plaque constituents and discovering new ones. STOMP can be applied to various biological samples including cell lines, primary cell cultures, ex vivo specimens, biopsy samples and fixed postmortem tissue.

    View details for DOI 10.7554/eLife.09579

    View details for PubMedID 26418743

  • Chemical Biology Strategies for Posttranslational Control of Protein Function CHEMISTRY & BIOLOGY Rakhit, R., Navarro, R., Wandless, T. J. 2014; 21 (9): 1238-1252
  • Chemical biology strategies for posttranslational control of protein function. Chemistry & biology Rakhit, R., Navarro, R., Wandless, T. J. 2014; 21 (9): 1238-1252


    A common strategy to understand a biological system is to selectively perturb it and observe its response. Although technologies now exist to manipulate cellular systems at the genetic and transcript level, the direct manipulation of functions at the protein level can offer significant advantages in precision, speed, and reversibility. Combining the specificity of genetic manipulation and the spatiotemporal resolution of light- and small molecule-based approaches now allows exquisite control over biological systems to subtly perturb a system of interest in vitro and in vivo. Conditional perturbation mechanisms may be broadly characterized by change in intracellular localization, intramolecular activation, or degradation of a protein-of-interest. Here we review recent advances in technologies for conditional regulation of protein function and suggest further areas of potential development.

    View details for DOI 10.1016/j.chembiol.2014.08.011

    View details for PubMedID 25237866

  • General Method for Regulating Protein Stability with Light ACS CHEMICAL BIOLOGY Bonger, K. M., Rakhit, R., Payumo, A. Y., Chen, J. K., Wandless, T. J. 2014; 9 (1): 111-115


    Post-translational regulation of protein abundance in cells is a powerful tool for studying protein function. Here, we describe a novel genetically encoded protein domain that is degraded upon exposure to nontoxic blue light. We demonstrate that fusion proteins containing this domain are rapidly degraded in cultured cells and in zebrafish upon illumination.

    View details for DOI 10.1021/cb400755b

    View details for Web of Science ID 000330098800012

    View details for PubMedID 24180414

  • Intracellular Context Affects Levels of a Chemically Dependent Destabilizing Domain PLOS ONE Sellmyer, M. A., Chen, L., Egeler, E. L., Rakhit, R., Wandless, T. J. 2012; 7 (9)


    The ability to regulate protein levels in live cells is crucial to understanding protein function. In the interest of advancing the tool set for protein perturbation, we developed a protein destabilizing domain (DD) that can confer its instability to a fused protein of interest. This destabilization and consequent degradation can be rescued in a reversible and dose-dependent manner with the addition of a small molecule that is specific for the DD, Shield-1. Proteins encounter different local protein quality control (QC) machinery when targeted to cellular compartments such as the mitochondrial matrix or endoplasmic reticulum (ER). These varied environments could have profound effects on the levels and regulation of the cytoplasmically derived DD. Here we show that DD fusions in the cytoplasm or nucleus can be efficiently degraded in mammalian cells; however, targeting fusions to the mitochondrial matrix or ER lumen leads to accumulation even in the absence of Shield-1. Additionally, we characterize the behavior of the DD with perturbants that modulate protein production, degradation, and local protein QC machinery. Chemical induction of the unfolded protein response in the ER results in decreased levels of an ER-targeted DD indicating the sensitivity of the DD to the degradation environment. These data reinforce that DD is an effective tool for protein perturbation, show that the local QC machinery affects levels of the DD, and suggest that the DD may be a useful probe for monitoring protein quality control machinery.

    View details for DOI 10.1371/journal.pone.0043297

    View details for Web of Science ID 000308738500009

    View details for PubMedID 22984418

  • Targeting of Monomer/Misfolded SOD1 as a Therapeutic Strategy for Amyotrophic Lateral Sclerosis JOURNAL OF NEUROSCIENCE Liu, H., Tjostheim, S., DaSilva, K., Taylor, D., Zhao, B., Rakhit, R., Brown, M., Chakrabartty, A., McLaurin, J., Robertson, J. 2012; 32 (26): 8791-8799


    There is increasing evidence that toxicity of mutant superoxide dismutase-1 (SOD1) in amyotrophic lateral sclerosis (ALS) is linked to its propensity to misfold and to aggregate. Immunotargeting of differently folded states of SOD1 has provided therapeutic benefit in mutant SOD1 transgenic mice. The specific region(s) of the SOD1 protein to which these immunization approaches target are, however, unknown. In contrast, we have previously shown, using a specific antibody [SOD1 exposed dimer interface (SEDI) antibody], that the dimer interface of SOD1 is abnormally exposed both in mutant SOD1 transgenic mice and in familial ALS cases associated with mutations in the SOD1 gene (fALS1). Here, we show the beneficial effects of an active immunization strategy using the SEDI antigenic peptide displayed on a branched peptide dendrimer to target monomer/misfolded in SOD1(G37R) and SOD1(G93A) mutant SOD1 transgenic mice. Immunization delayed disease onset and extended disease duration, with survival times increased by an average of 40 d in SOD1(G37R) mice. Importantly, this immunization strategy favored a Th2 immune response, thereby precluding deleterious neuroinflammatory effects. Furthermore, the beneficial effects of immunization correlated with a reduction in accumulation of both monomer/misfolded and oligomeric SOD1 species in the spinal cord, the intended targets of the immunization strategy. Our results support that SOD1 misfolding/aggregation plays a central role in SOD1-linked ALS pathogenesis and identifies monomeric/misfolded SOD1 as a therapeutic target for SOD1-related ALS.

    View details for DOI 10.1523/JNEUROSCI.5053-11.2012

    View details for Web of Science ID 000305890700005

    View details for PubMedID 22745481

  • Ligand-switchable Substrates for a Ubiquitin-Proteasome System JOURNAL OF BIOLOGICAL CHEMISTRY Egeler, E. L., Urner, L. M., Rakhit, R., Liu, C. W., Wandless, T. J. 2011; 286 (36): 31328-31336


    Cellular maintenance of protein homeostasis is essential for normal cellular function. The ubiquitin-proteasome system (UPS) plays a central role in processing cellular proteins destined for degradation, but little is currently known about how misfolded cytosolic proteins are recognized by protein quality control machinery and targeted to the UPS for degradation in mammalian cells. Destabilizing domains (DDs) are small protein domains that are unstable and degraded in the absence of ligand, but whose stability is rescued by binding to a high affinity cell-permeable ligand. In the work presented here, we investigate the biophysical properties and cellular fates of a panel of FKBP12 mutants displaying a range of stabilities when expressed in mammalian cells. Our findings correlate observed cellular instability to both the propensity of the protein domain to unfold in vitro and the extent of ubiquitination of the protein in the non-permissive (ligand-free) state. We propose a model in which removal of stabilizing ligand causes the DD to unfold and be rapidly ubiquitinated by the UPS for degradation at the proteasome. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable UPS substrate unlike other methods currently used to interrogate protein quality control, providing tunable control of degradation rates.

    View details for DOI 10.1074/jbc.M111.264101

    View details for Web of Science ID 000294487500028

    View details for PubMedID 21768107

  • Evaluation of FKBP and DHFR based destabilizing domains in Saccharomyces cerevisiae BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Rakhit, R., Edwards, S. R., Iwamoto, M., Wandless, T. J. 2011; 21 (17): 4965-4968


    Two orthogonal destabilizing domains have been developed based on mutants of human FKBP12 as well as bacterial DHFR and these engineered domains have been used to control protein concentration in a variety of contexts in vitro and in vivo. FKBP12 based destabilizing domains cannot be rescued in the yeast Saccharomyces cerevisiae; ecDHFR based destabilizing domains are not degraded as efficiently in S. cerevisiae as in mammalian cells or Plasmodium, but provide a starting point for the development of domains with increased signal-to-noise in S. cerevisiae.

    View details for DOI 10.1016/j.bmcl.2011.06.006

    View details for Web of Science ID 000293884100006

    View details for PubMedID 21741238

  • An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS NATURE MEDICINE Rakhit, R., Robertson, J., Vande Velde, C., Horne, P., Ruth, D. M., Griffin, J., Cleveland, D. W., Cashman, N. R., Chakrabartty, A. 2007; 13 (6): 754-759


    Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.

    View details for DOI 10.1038/nm1559

    View details for Web of Science ID 000247084300041

    View details for PubMedID 17486090

  • Structure, folding, and misfolding of Cu,Zn superoxide dismutase in amyotrophic lateral sclerosis BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE Rakhit, R., Chakrabartty, A. 2006; 1762 (11-12): 1025-1037


    Fourteen years after the discovery that mutations in Cu, Zn superoxide dismutase (SOD1) cause a subset of familial amyotrophic lateral sclerosis (fALS), the mechanism by which mutant SOD1 exerts toxicity remains unknown. The two principle hypotheses are (a) oxidative damage stemming from aberrant SOD1 redox chemistry, and (b) misfolding of the mutant protein. Here we review the structure and function of wild-type SOD1, as well as the changes to the structure and function in mutant SOD1. The relative merits of the two hypotheses are compared and a common unifying principle is outlined. Lastly, the potential for therapies targeting SOD1 misfolding is discussed.

    View details for DOI 10.1016/j.bbadis.2006.05.004

    View details for Web of Science ID 000242926600007

    View details for PubMedID 16814528

  • Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis JOURNAL OF BIOLOGICAL CHEMISTRY Rakhit, R., Crow, J. P., Lepock, J. R., Kondejewski, L. H., Cashman, N. R., Chakrabartty, A. 2004; 279 (15): 15499-15504


    Proteinacious intracellular aggregates in motor neurons are a key feature of both sporadic and familial amyotrophic lateral sclerosis (ALS). These inclusion bodies are often immunoreactive for Cu,Zn-superoxide dismutase (SOD1) and are implicated in the pathology of ALS. On the basis of this and a similar clinical presentation of symptoms in the familial (fALS) and sporadic forms of ALS, we sought to investigate the possibility that there exists a common disease-related aggregation pathway for fALS-associated mutant SODs and wild type SOD1. We have previously shown that oxidation of fALS-associated mutant SODs produces aggregates that have the same morphological, structural, and tinctorial features as those found in SOD1 inclusion bodies in ALS. Here, we show that oxidative damage of wild type SOD at physiological concentrations ( approximately 40 microm) results in destabilization and aggregation in vitro. Oxidation of either mutant or wild type SOD1 causes the enzyme to dissociate to monomers prior to aggregation. Only small changes in secondary and tertiary structure are associated with monomer formation. These results indicate a common aggregation prone monomeric intermediate for wild type and fALS-associated mutant SODs and provides a link between sporadic and familial ALS.

    View details for DOI 10.1074/jbc.M313295200

    View details for Web of Science ID 000220594700121

    View details for PubMedID 14734542

  • Oxidation-induced misfolding and agagaregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis JOURNAL OF BIOLOGICAL CHEMISTRY Rakhit, R., Cunningham, P., Furtos-Matei, A., Dahan, S., Qi, X. F., Crow, J. P., Cashman, N. R., Kondejewski, L. H., Chakrabartty, A. 2002; 277 (49): 47551-47556


    The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS.

    View details for DOI 10.1074/jbc.M207356200

    View details for Web of Science ID 000179663700093

    View details for PubMedID 12356748

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