Showing papers by "Vamsi K. Mootha published in 2022"

CRISPR-free base editors with enhanced activity and expanded targeting scope in mitochondrial and nuclear DNA

Abstract: The all-protein cytosine base editor DdCBE uses TALE proteins and a double-stranded DNA-specific cytidine deaminase (DddA) to mediate targeted C•G-to-T•A editing. To improve editing efficiency and overcome the strict TC sequence-context constraint of DddA, we used phage-assisted non-continuous and continuous evolution to evolve DddA variants with improved activity and expanded targeting scope. Compared to canonical DdCBEs, base editors with evolved DddA6 improved mitochondrial DNA (mtDNA) editing efficiencies at TC by 3.3-fold on average. DdCBEs containing evolved DddA11 offered a broadened HC (H = A, C or T) sequence compatibility for both mitochondrial and nuclear base editing, increasing average editing efficiencies at AC and CC targets from less than 10% for canonical DdCBE to 15-30% and up to 50% in cell populations sorted to express both halves of DdCBE. We used these evolved DdCBEs to efficiently install disease-associated mtDNA mutations in human cells at non-TC target sites. DddA6 and DddA11 substantially increase the effectiveness and applicability of all-protein base editing.

Medical thoracoscopy for undiagnosed pleural effusions: experience from a tertiary care hospital in north India.

Abstract: BACKGROUND AND AIMS Medical thoracoscopy, also called pleuroscopy, has received renewed interest in the recent past for diagnostic as well as therapeutic uses. In this study, we describe our experience with thoracoscopy for undiagnosed pleural effusions. METHODS In a retrospective analysis of thoracoscopic procedures we performed between January 2007 and December 2008, yield of thoracoscopic pleural biopsy for achieving a diagnosis in undiagnosed pleural effusions, defined as pleural effusions with adenosine deaminase (ADA) levels less than 70 IU/L and negative pleural fluid cytology for malignancy on three occasions was evaluated. Complications of thoracoscopy were also analysed. RESULTS Overall diagnostic yield of thoracoscopic pleural biopsy was 74.3% in patients with undiagnosed pleural effusions. Pleural malignancy was diagnosed in 48.6% of patients. There was only one case of mesothelioma and the rest were due to pleural metastasis. Lung cancer and breast cancer were the most common sites of primary malignancy. Tuberculosis was diagnosed with pleural biopsy in 22.8% of patients. We had low complication rate after thoracoscopy. Only two cases of empyema were observed. CONCLUSION Medical thoracoscopy is a safe procedure and has good diagnostic yield in patients with undiagnosed pleural effusions.

Combinatorial GxGxE CRISPR screen identifies SLC25A39 in mitochondrial glutathione transport linking iron homeostasis to OXPHOS

Abstract: Abstract The SLC25 carrier family consists of 53 transporters that shuttle nutrients and co-factors across mitochondrial membranes. The family is highly redundant and their transport activities coupled to metabolic state. Here, we use a pooled, dual CRISPR screening strategy that knocks out pairs of transporters in four metabolic states — glucose, galactose, OXPHOS inhibition, and absence of pyruvate — designed to unmask the inter-dependence of these genes. In total, we screen 63 genes in four metabolic states, corresponding to 2016 single and pair-wise genetic perturbations. We recover 19 gene-by-environment (GxE) interactions and 9 gene-by-gene (GxG) interactions. One GxE interaction hit illustrates that the fitness defect in the mitochondrial folate carrier (SLC25A32) KO cells is genetically buffered in galactose due to a lack of substrate in de novo purine biosynthesis. GxG analysis highlights a buffering interaction between the iron transporter SLC25A37 (A37) and the poorly characterized SLC25A39 (A39). Mitochondrial metabolite profiling, organelle transport assays, and structure-guided mutagenesis identify A39 as critical for mitochondrial glutathione (GSH) import. Functional studies reveal that A39-mediated glutathione homeostasis and A37-mediated mitochondrial iron uptake operate jointly to support mitochondrial OXPHOS. Our work underscores the value of studying family-wide genetic interactions across different metabolic environments.

Discovery and molecular basis of subtype-selective cyclophilin inhibitors

Abstract: Although cyclophilins are attractive targets for probing biology and therapeutic intervention, no subtype-selective cyclophilin inhibitors have been described. We discovered novel cyclophilin inhibitors from the in vitro selection of a DNA-templated library of 256,000 drug-like macrocycles for cyclophilin D (CypD) affinity. Iterated macrocycle engineering guided by ten X-ray co-crystal structures yielded potent and selective inhibitors (half maximal inhibitory concentration (IC50) = 10 nM) that bind the active site of CypD and also make novel interactions with non-conserved residues in the S2 pocket, an adjacent exo-site. The resulting macrocycles inhibit CypD activity with 21- to >10,000-fold selectivity over other cyclophilins and inhibit mitochondrial permeability transition pore opening in isolated mitochondria. We further exploited S2 pocket interactions to develop the first cyclophilin E (CypE)-selective inhibitor, which forms a reversible covalent bond with a CypE S2 pocket lysine, and exhibits 30- to >4,000-fold selectivity over other cyclophilins. These findings reveal a strategy to generate isoform-selective small-molecule cyclophilin modulators, advancing their suitability as targets for biological investigation and therapeutic development.

Congenital hypermetabolism and uncoupled oxidative phosphorylation

Abstract: Summary We describe the case of identical twin boys who presented with low body weight despite excessive caloric intake. An evaluation of their fibroblasts showed elevated oxygen consumption and decreased mitochondrial membrane potential. Exome analysis revealed a de novo heterozygous variant in ATP5F1B, which encodes the β subunit of mitochondrial ATP synthase (also called complex V). In yeast, mutations affecting the same region loosen coupling between the proton motive force and ATP synthesis, resulting in high rates of mitochondrial respiration. Expression of the mutant allele in human cell lines recapitulates this phenotype. These data support an autosomal dominant mitochondrial uncoupling syndrome with hypermetabolism.

METTL17 is an Fe-S cluster checkpoint for mitochondrial translation

Abstract: Friedreich’s ataxia (FA) is the most common monogenic mitochondrial disease. FA is caused by a depletion of the mitochondrial protein frataxin (FXN), an iron-sulfur (Fe-S) cluster biogenesis factor. To better understand the cellular consequences of FA, we performed quantitative proteome profiling of human cells depleted for FXN. Nearly every known Fe-S cluster-containing protein was depleted in the absence of FXN, indicating that as a rule, cluster binding confers stability to Fe-S proteins. Proteomic and genetic interaction mapping identified impaired mitochondrial translation downstream of FXN loss, and specifically highlighted the methyltransferase-like protein METTL17 as a candidate effector. Using comparative sequence analysis, mutagenesis, biochemistry and cryogenic electron microscopy we show that METTL17 binds to the mitoribosomal small subunit during late assembly and harbors a previously unrecognized [Fe4S4]2+ cluster required for its stability on the mitoribosome. Notably, METTL17 overexpression rescued the mitochondrial translation and bioenergetic defects, but not the cellular growth, of FXN null cells. Our data suggest that METTL17 serves as an Fe-S cluster checkpoint: promoting the translation and assembly of Fe-S cluster rich OXPHOS proteins only when Fe-S cluster levels are replete.

Effectors enabling adaptation to mitochondrial complex I loss in Hürthle cell carcinoma

Abstract: Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is genetically characterized by complex I mitochondrial DNA mutations and widespread chromosomal losses. Here, we utilize RNA-seq and metabolomics to identify candidate molecular effectors activated by these genetic drivers. We find glutathione biosynthesis, amino acid metabolism, mitochondrial unfolded protein response, and lipid peroxide scavenging, a safeguard against ferroptosis, to be increased in HCC. A CRISPR-Cas9 knockout screen in a new HCC model reveals which pathways are key for fitness, and highlights loss of GPX4, a defense against ferroptosis, as a strong liability. Rescuing complex I redox activity with the yeast NADH dehydrogenase (NDI1) in HCC cells diminishes ferroptosis sensitivity, while inhibiting complex I in normal thyroid cells augments ferroptosis induction. Our work demonstrates unmitigated lipid peroxide stress to be an HCC vulnerability that is mechanistically coupled to the genetic loss of mitochondrial complex I activity. Significance Oncocytic (Hürthle cell) carcinoma of the thyroid (HCC) is a unique tumor with a remarkable accumulation of mitochondria. HCC harbors unique genetic alterations, including mitochondrial DNA (mtDNA) mutations in complex I genes and widespread loss-of-heterozygosity in the nuclear DNA. With less favorable clinical outcomes, new therapies for HCC are needed, especially since these tumors show intrinsic resistance to radioactive iodine, which is one of the main treatments for metastatic well-differentiated thyroid cancer. An absence of authentic HCC cell lines and animal models has hindered the mechanistic understanding of this disease and slowed therapeutic progress. In this study, we describe the transcriptomic and metabolomic landscape of HCC and present new HCC models that recapitulate key mtDNA and nuclear DNA alterations. A targeted CRISPR-Cas9 knockout screen in an HCC cell line highlights the molecular programs nominated by our -omics profiling that are required for cell fitness. This screen suggests that lipid peroxide scavenging, a defense system against an iron-dependent form of cell death known as ferroptosis, is a vulnerability in HCC that is coupled to complex I loss, and that targeting this pathway may help patients with HCC.

A genetically encoded system for oxygen generation in living cells

Abstract: Significance Oxygen is one of the most important molecules in living systems, playing a key role in energy metabolism, cellular signaling, and disease. At present, we have few if any ways to manipulate molecular oxygen in living cells with high spatiotemporal control. Here, we introduce a genetic strategy for generating oxygen inside human cells, by simultaneously expressing a transporter and a bacterial enzyme. Together, these proteins promote the uptake of chlorite into the cell and enzymatically produce oxygen. We call this genetic technology SupplemeNtal Oxygen Released from ChLorite (SNORCL). This technology will allow investigation of the effects of short, local pulses of oxygen in cells and tissues. Optimized versions of the technology could have direct medical applications.

A natural fusion of flavodiiron, rubredoxin, and NADH:rubredoxin oxidoreductase domains is the highly efficient water-forming oxidase of T. vaginalis

Abstract: Microaerophilic pathogens such as Giardia lamblia and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain the intracellular redox balance. This oxygen consumption is a result of the H2O-forming NADH oxidase activity of two distinct flavin-containing systems: H2O-forming NADH oxidases (NOXes) and multicomponent flavodiiron proteins (FDPs). Both systems are not membrane-bound and recycle NADH into oxidized NAD+ while simultaneously removing O2 from the local environment, making them crucial for the survival of human microaerophilic pathogens. In this study, using bioinformatic and biochemical analysis, we show that T. vaginalis lacks a NOX-like enzyme, and instead harbors three proteins that are very close in their amino acid sequence and represent a natural fusion between N-terminal FDP, central rubredoxin and C-terminal NADH:rubredoxin oxidoreductase domains. We demonstrate that this natural fusion protein with fully populated flavin redox centers unlike a “stand-alone” FDP (also present in T. vaginalis), directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of O2 to water within a single polypeptide and with an extremely high turnover. Using single particle electron cryo-microscopy (cryo-EM) we present structural insight into the spatial organization of the FDP core within this multidomain fusion protein. Our studies represent an important addition to our understanding of systems that allow human protozoan parasites to maintain their optimal redox balance and survive transient exposure to oxic conditions.