A5033184755- Metalloenzymes and iron-sulfur proteins
- CRISPR and Genetic Engineering
- Metal-Catalyzed Oxygenation Mechanisms
- CAR-T cell therapy research
- Metal complexes synthesis and properties
- RNA regulation and disease
- RNA and protein synthesis mechanisms
- Pluripotent Stem Cells Research
- Hemoglobinopathies and Related Disorders
- Prenatal Screening and Diagnostics
- Trace Elements in Health
- Chemical Synthesis and Analysis
- RNA Interference and Gene Delivery
- RNA modifications and cancer
- Enzyme Structure and Function
- bioluminescence and chemiluminescence research
- Virus-based gene therapy research
- DNA and Nucleic Acid Chemistry
- Mitochondrial Function and Pathology
- Biochemical and Molecular Research
- Protein Structure and Dynamics
- Advanced biosensing and bioanalysis techniques
- Endoplasmic Reticulum Stress and Disease
- Folate and B Vitamins Research
- Viral Infections and Immunology Research
Broad Institute
2022-2023
Editas Medicine (United States)
2018-2023
Howard Hughes Medical Institute
2015-2016
Harvard University
2014-2016
University of California, San Diego
2009-2014
Hebrew University of Jerusalem
2010
Significance The therapeutic potential of protein-based genome editing is dependent on the delivery proteins to appropriate intracellular targets. Here we report that combining bioreducible lipid nanoparticles and negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives self-assembly for potent protein editing. design lipids facilitates degradation inside cells in response reductive environment, enhancing endosome escape protein. In addition, modulation...
Though AsCas12a fills a crucial gap in the current genome editing toolbox, it exhibits relatively poor efficiency, restricting its overall utility. Here we isolate an engineered variant, "AsCas12a Ultra", that increased efficiency to nearly 100% at all sites examined HSPCs, iPSCs, T cells, and NK cells. We show Ultra maintains high on-target specificity thereby mitigating risk for off-target making ideal complex therapeutic applications. achieved simultaneous targeting of three clinically...
MitoNEET (mNT) is an outer mitochondrial membrane target of the thiazolidinedione diabetes drugs with a unique fold and labile [2Fe-2S] cluster. The rare 1-His 3-Cys coordination mNT’s leads to cluster lability that strongly dependent on presence single histidine ligand (His87). These properties mNT are similar known shuttle proteins. Here we investigated whether capable transfer acceptor protein(s). Facile observed between oxidized apo-ferredoxin (a-Fd) using UV-VIS spectroscopy...
Mutations in Atp2b2, an outer hair cell gene, cause dominant hearing loss humans. Using a mouse model Atp2b2
The NEET family is a newly discovered group of proteins involved in diverse array biological processes, including autophagy, apoptosis, aging, diabetes, and reactive oxygen homeostasis. They form novel structure, the fold, which two protomers intertwine to two-domain motif, cap, unique redox-active labile 2Fe-2S cluster binding domain. To accelerate functional study proteins, as well examine whether they have an evolutionarily conserved role, we identified characterized plant protein. Here,...
MitoNEET is a small mitochondrial protein that has been identified recently as target for the thiazolidinedione (TZD) class of diabetes drugs. also binds unique three-His- and one-Cys-ligated [2Fe-2S] cluster. Here we use film voltammetry (PFV) means to probe redox properties mitoNEET demonstrate direct impact TZD drug binding upon chemistry FeS When TZDs bind, midpoint potential at pH 7 lowered by more than 100 mV, shifting from ∼0 −100 mV. In contrast, His87Cys mutant negates ability...
Significance Misregulation of cell growth and proliferation leads to the onset various diseases, including cancer. Two proteins crucial for proper cellular control that were recently shown affect are Bcl-2, well-known its role in programmed death, newly identified iron-sulfur protein NAF-1, localized near mitochondrial outer membrane. In this report, we use a strategy utilizing combination experimental computational techniques provides valuable information enable us determine molecular...
Multiplexed genome editing with DNA endonucleases has broad application, including for cellular therapies, but chromosomal translocations, natural byproducts of inducing simultaneous genomic breaks, have not been explored in detail. Here we apply various CRISPR-Cas nucleases to edit the T cell receptor alpha and beta 2 microglobulin genes human primary cells comprehensively evaluate frequency stability resulting translocations. A thorough translocation analysis using three orthogonal methods...
MitoNEET is a newly discovered mitochondrial protein and target of the TZD class antidiabetes drugs. homodimeric with each protomer binding [2Fe-2S] center through rare 3-Cys 1-His coordination geometry. Both fold centers suggest that it could have novel properties compared to other known proteins. We tested robustness mitoNEET mutation range over which redox potential (EM) be tuned. found tolerate an array mutations modified EM ∼700 mV, largest engineered in FeS and, importantly, spans...
Nutrient-deprivation autophagy factor-1 (NAF-1) (synonyms: Cisd2, Eris, Miner1, and Noxp70) is a [2Fe-2S] cluster protein immune-detected both in endoplasmic reticulum (ER) mitochondrial outer membrane. It was implicated human pathology (Wolfram Syndrome 2) BCL-2 mediated antagonization of Beclin 1-dependent depression ER calcium stores. To gain insights about NAF-1 functions, we investigated the biochemical properties its 2Fe-2S sensitivity those to small molecules. The structure soluble...
MitoNEET (mNT) is the founding member of recently discovered CDGSH family [2Fe-2S] proteins capable cluster transfer to apo-acceptor proteins. It a target thiazolidinedione (TZD) class anti-diabetes drugs whose binding modulate both electron and properties. The in mNT destabilized upon NADPH, which leads loss solution environment. Because transferring clusters proteins, we sought determine whether NADPH also affects transfer. We show that inhibits an protein with inhibition constant (K(i))...
Regulation of protein function via cracking, or local unfolding and refolding substructures, is becoming a widely recognized mechanism functional control. Oftentimes, cracking events are localized to secondary tertiary structure interactions between domains that control the optimal position for catalysis and/or formation complexes. Small changes in free energy associated with ligand binding, phosphorylation, etc., can tip balance provide regulatory switch. However, understanding factors...
MitoNEET is the only identified Fe–S protein localized to outer mitochondrial membrane and a 1.5 Å resolution X-ray analysis has revealed unique structure [Paddock et al. (2007), Proc. Natl Acad. Sci. USA, 104, 14342–14347]. The 2Fe–2S cluster bound with 3Cys–1His coordination which defines new class of proteins. hallmark feature this single noncysteine ligand His87, when replaced by Cys decreases redox potential (Em) ∼300 mV increases stability around sixfold. Unexpectedly, pH dependence...
Metalloproteins (MPs) comprise one-third of all known protein structures. This diverse set proteins contain a plethora unique inorganic moieties capable performing chemistry that would otherwise be impossible using only the amino acids found in nature. Most well-studied MPs are generally viewed as being very rigid structure, and it is widely thought properties metal centers primarily determined by small fraction make up local environment. Here we examine both theoretically experimentally...
Background: Induction of fetal hemoglobin (HbF) is an attractive approach to ameliorate disease symptoms in beta hemoglobinopathies. Gene editing by zinc finger or CRISPR‐Cas nucleases disrupt BCL11A expression, a known mediator globin silencing, via targeting erythroid‐enhancer, one reactivate expression. Alternatively, the genetics hereditary persistence (HPFH) suggest that disruption certain cis‐regulatory elements beta‐globin locus may also be viable strategy. A previous lenti‐CRISPR...