Publications by authors named "Elliot Dine"

8 Publications

  • Page 1 of 1

Positive feedback between the T cell kinase Zap70 and its substrate LAT acts as a clustering-dependent signaling switch.

Cell Rep 2021 Jun;35(12):109280

Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA; IRCC International Research Collaboration Center, National Institutes of Natural Sciences, 4-3-13 Toranomon, Minato-ku, Tokyo 105-0001, Japan. Electronic address:

Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state.
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http://dx.doi.org/10.1016/j.celrep.2021.109280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8292983PMC
June 2021

Mutations Outside the Ure2 Amyloid-Forming Region Disrupt [URE3] Prion Propagation and Alter Interactions with Protein Quality Control Factors.

Mol Cell Biol 2020 10 13;40(21). Epub 2020 Oct 13.

Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA

The yeast prion [URE3] propagates as a misfolded amyloid form of the Ure2 protein. Propagation of amyloid-based yeast prions requires protein quality control (PQC) factors, and altering PQC abundance or activity can cure cells of prions. Yeast antiprion systems composed of PQC factors act at normal abundance to restrict establishment of the majority of prion variants that arise While these systems are well described, how they or other PQC factors interact with prion proteins remains unclear. To gain insight into such interactions, we identified mutations outside the Ure2 prion-determining region that destabilize [URE3]. Despite residing in the functional domain, 16 of 17 mutants retained Ure2 activity. Four characterized mutations caused rapid loss of [URE3] yet allowed [URE3] to propagate under prion-selecting conditions. Two sensitized [URE3] to Btn2, Cur1, and Hsp42, but in different ways. Two others reduced amyloid formation Of these, one impaired prion replication and the other apparently impaired transmission. Thus, widely dispersed sites outside a prion's amyloid-forming region can contribute to prion character, and altering such sites can disrupt prion propagation by altering interactions with PQC factors.
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http://dx.doi.org/10.1128/MCB.00294-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556844PMC
October 2020

Light-based control of metabolic flux through assembly of synthetic organelles.

Nat Chem Biol 2019 06 13;15(6):589-597. Epub 2019 May 13.

Department of Molecular Biology, Princeton University, Princeton, NJ, USA.

To maximize a desired product, metabolic engineers typically express enzymes to high, constant levels. Yet, permanent pathway activation can have undesirable consequences including competition with essential pathways and accumulation of toxic intermediates. Faced with similar challenges, natural metabolic systems compartmentalize enzymes into organelles or post-translationally induce activity under certain conditions. Here we report that optogenetic control can be used to extend compartmentalization and dynamic control to engineered metabolisms in yeast. We describe a suite of optogenetic tools to trigger assembly and disassembly of metabolically active enzyme clusters. Using the deoxyviolacein biosynthesis pathway as a model system, we find that light-switchable clustering can enhance product formation six-fold and product specificity 18-fold by decreasing the concentration of intermediate metabolites and reducing flux through competing pathways. Inducible compartmentalization of enzymes into synthetic organelles can thus be used to control engineered metabolic pathways, limit intermediates and favor the formation of desired products.
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http://dx.doi.org/10.1038/s41589-019-0284-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6755918PMC
June 2019

Protein Phase Separation Provides Long-Term Memory of Transient Spatial Stimuli.

Cell Syst 2018 06 30;6(6):655-663.e5. Epub 2018 May 30.

Department of Molecular Biology, Princeton University, Washington Road, Princeton, NJ 08544, USA. Electronic address:

Protein/RNA clusters arise frequently in spatially regulated biological processes, from the asymmetric distribution of P granules and PAR proteins in developing embryos to localized receptor oligomers in migratory cells. This co-occurrence suggests that protein clusters might possess intrinsic properties that make them a useful substrate for spatial regulation. Here, we demonstrate that protein droplets show a robust form of spatial memory, maintaining the spatial pattern of an inhibitor of droplet formation long after it has been removed. Despite this persistence, droplets can be highly dynamic, continuously exchanging monomers with the diffuse phase. We investigate the principles of biophysical spatial memory in three contexts: a computational model of phase separation; a novel optogenetic system where light can drive rapid, localized dissociation of liquid-like protein droplets; and membrane-localized signal transduction from clusters of receptor tyrosine kinases. Our results suggest that the persistent polarization underlying many cellular and developmental processes could arise through a simple biophysical process, without any additional biochemical feedback loops.
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http://dx.doi.org/10.1016/j.cels.2018.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023754PMC
June 2018

Optogenetic Reconstitution for Determining the Form and Function of Membraneless Organelles.

Biochemistry 2018 05 26;57(17):2432-2436. Epub 2018 Jan 26.

Department of Molecular Biology , Princeton University , Princeton , New Jersey 08544 , United States.

It has recently become clear that large-scale macromolecular self-assembly is a rule, rather than an exception, of intracellular organization. A growing number of proteins and RNAs have been shown to self-assemble into micrometer-scale clusters that exhibit either liquid-like or gel-like properties. Given their proposed roles in intracellular regulation, embryo development, and human disease, it is becoming increasingly important to understand how these membraneless organelles form and to map their functional consequences for the cell. Recently developed optogenetic systems make it possible to acutely control cluster assembly and disassembly in live cells, driving the separation of proteins of interest into liquid droplets, hydrogels, or solid aggregates. Here we propose that these approaches, as well as their evolution into the next generation of optogenetic biophysical tools, will allow biologists to determine how the self-assembly of membraneless organelles modulates diverse biochemical processes.
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http://dx.doi.org/10.1021/acs.biochem.7b01173DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5972035PMC
May 2018

A Genetic Incompatibility Accelerates Adaptation in Yeast.

PLoS Genet 2015 Jul 31;11(7):e1005407. Epub 2015 Jul 31.

Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, United States of America.

During mismatch repair (MMR) MSH proteins bind to mismatches that form as the result of DNA replication errors and recruit MLH factors such as Mlh1-Pms1 to initiate excision and repair steps. Previously, we identified a negative epistatic interaction involving naturally occurring polymorphisms in the MLH1 and PMS1 genes of baker's yeast. Here we hypothesize that a mutagenic state resulting from this negative epistatic interaction increases the likelihood of obtaining beneficial mutations that can promote adaptation to stress conditions. We tested this by stressing yeast strains bearing mutagenic (incompatible) and non-mutagenic (compatible) mismatch repair genotypes. Our data show that incompatible populations adapted more rapidly and without an apparent fitness cost to high salt stress. The fitness advantage of incompatible populations was rapid but disappeared over time. The fitness gains in both compatible and incompatible strains were due primarily to mutations in PMR1 that appeared earlier in incompatible evolving populations. These data demonstrate a rapid and reversible role (by mating) for genetic incompatibilities in accelerating adaptation in eukaryotes. They also provide an approach to link experimental studies to observational population genomics.
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http://dx.doi.org/10.1371/journal.pgen.1005407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4521705PMC
July 2015

The transmission interface of the Saccharomyces cerevisiae multidrug transporter Pdr5: Val-656 located in intracellular loop 2 plays a major role in drug resistance.

Antimicrob Agents Chemother 2013 Feb 17;57(2):1025-34. Epub 2012 Dec 17.

Department of Biology, The Catholic University of America, Washington, District of Columbia, USA.

Pdr5 is a major ATP-binding cassette (ABC) multidrug transporter regarded as the founding member of a fungal subfamily of clinically significant efflux pumps. When these proteins are overexpressed, they confer broad-spectrum ultraresistance. To better understand the evolution of these proteins under selective pressure, we exposed a Saccharomyces cerevisiae yeast strain already overexpressing Pdr5 to a lethal concentration of cycloheximide. This approach gave mutations that confer greater resistance to a subset of transport substrates. One of these mutations, V656L, is located in intracellular loop 2 (ICL2), a region predicted by structural studies with several other ABC transporters to play a critical role in the transmission interface between the ATP hydrolysis and drug transport domains. We show that this mutation increases drug resistance, possibly by altering the efficiency with which the energy from ATP hydrolysis is used for transport. Val-656 is a conserved residue, and an alanine substitution creates a nearly null phenotype for drug transport as well as reduced ATPase activity. We posit that despite its unusually small size, ICL2 is part of the transmission interface, and that alterations in this pathway can increase or decrease resistance to a broad spectrum of drugs.
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http://dx.doi.org/10.1128/AAC.02133-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3553689PMC
February 2013

The signaling interface of the yeast multidrug transporter Pdr5 adopts a cis conformation, and there are functional overlap and equivalence of the deviant and canonical Q-loop residues.

Biochemistry 2010 Jun;49(21):4440-9

Department of Biology, The Catholic University of America, Washington, DC 20064, USA.

ABC transporters are polytopic proteins. ATP hydrolysis and substrate transport take place in separate domains, and these activities must be coordinated through a signal interface. We previously characterized a mutation (S558Y) in the yeast multidrug transporter Pdr5 that uncouples ATP hydrolysis and drug transport. To characterize the transmission interface, we used a genetic screen to isolate second-site mutations of S558Y that restore drug transport. We recovered suppressors that restore drug resistance; their locations provide functional evidence for an interface in the cis rather than the trans configuration indicated by structural and cross-linking studies of bacterial and eukaryotic efflux transporters. One mutation, E244G, defines the Q-loop of the deviant portion of NBD1, which is the hallmark of this group of fungal transporters. When moved to an otherwise wild-type background, this mutation and its counterpart in the canonical ATP-binding site Q951G show a similar reduction in drug resistance and in the very high basal-level ATP hydrolysis characteristic of Pdr5. A double E244G, Q951G mutant is considerably more drug sensitive than either of the single mutations. Surprisingly, then, the deviant and canonical Q-loop residues are functionally overlapping and equivalent in a strikingly asymmetric ABC transporter.
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http://dx.doi.org/10.1021/bi100394jDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2954458PMC
June 2010
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