Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis in<i>Candida albicans</i>

Weissman, Ziva; Pinsky, Mariel; Donegan, Rebecca K.; Reddi, Amit R.; Kornitzer, Daniel

doi:10.1101/2020.07.26.221606

Cited by 2 publications

(2 citation statements)

References 56 publications

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“…Labile Heme Measurements. Labile heme was measured using a previously reported genetically encoded heme sensor, HS1 29,[37][38][39]43,45,46 . HS1 was expressed using the pcDNA3.1 plasmid and driven by the CMV promoter, as previously described 38,43 .…”

Section: Heme Affinity Chromatography (Hac)mentioning

confidence: 99%

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Kim

Moore

Mestre-Fos

et al. 2022

Preprint

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Heme b (iron protoporphyrin IX) plays important roles in biology as a metallocofactor and signaling molecule. However, the targets of heme signaling and the network of proteins that mediate the exchange of heme from sites of synthesis or uptake to heme dependent or regulated proteins are poorly understood. Herein, we describe a quantitative mass spectrometry-based chemoproteomics strategy to identify exchange labile hemoproteins in human embryonic kidney HEK293 cells that may be relevant to heme signaling and trafficking. The strategy involves depleting endogenous heme with the heme biosynthetic inhibitor succinylacetone (SA), leaving putative heme binding proteins in their apo-state, followed by the capture of those proteins using hemin-agarose resin and finally elution and identification by mass spectrometry. By identifying only those proteins that interact with high specificity to hemin-agarose relative to control beaded agarose in a SA-dependent manner, we have expanded the number of proteins and ontologies that may be involved in binding and buffering labile heme or are targets of heme signaling. Notably, these include proteins involved in chromatin remodeling, DNA damage response, RNA splicing, cytoskeletal organization and vesicular trafficking, many of which have been associated with heme through complimentary studies published recently. Taken together, these results provide support for the emerging role for heme in an expanded set of cellular processes from genome integrity to protein trafficking and beyond.

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Section: Heme Affinity Chromatography (Hac)mentioning

confidence: 99%

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Kim

Moore

Mestre-Fos

et al. 2022

Preprint

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“…However, compared with the HEME-T4 strain (C41ΔhemA strain harboring pCDF-P J23117 -ChuA), the capacity for heme uptake was reduced in the HEME-T7 strain (Figure 3b). The genetically encoded ratiometric fluorescent sensors [32,33] detected the capacity of heme uptake, and 2.5 nm of hemin was imported into the cells through ChuA in the HEME-T4 strain when 10 mg L −1 hemin was supplemented (Figure S2, Supporting Information).…”

Section: Improving Heme Supply By Introducing Heterologous Heme Impor...mentioning

confidence: 99%

Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

Zhou

et al. 2022

Advanced Science

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By exploiting versatile P450 enzymes, whole-cell biocatalysis can be performed to synthesize valuable compounds in Escherichia coli. However, the insufficient supply of heme limits the whole-cell P450 biocatalytic activity. Here a strategy for improving intracellular heme biosynthesis to enhance the catalytic efficiencies of P450s is reported. After comparing the effects of improving heme transport and biosynthesis on P450 activities, intracellular heme biosynthesis is optimized through the integrated expression of necessary synthetic genes at proper ratios and the assembly of rate-limiting enzymes using DNA-guided scaffolds. The intracellular heme level is fine-tuned by the combined use of mutated heme-sensitive biosensors and small regulatory RNA systems. The catalytic efficiencies of three different P450s, BM3, sca-2, and CYP105D7, are enhanced through fine-tuning heme biosynthesis for the synthesis of hydroquinone, pravastatin, and 7,3′,4′-trihydroxyisoflavone as example products of chemical intermediate, drug, and natural product, respectively. This strategy of fine-tuned heme biosynthesis will be generally useful for developing whole-cell biocatalysts involving hemoproteins.

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Using genetically encoded heme sensors to probe the mechanisms of heme uptake and homeostasis inCandida albicans

Cited by 2 publications

References 56 publications

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Depletion Assisted Hemin Affinity (DAsHA) Proteomics Reveals an Expanded Landscape of Heme Binding Proteins

Whole‐Cell P450 Biocatalysis Using Engineered Escherichia coli with Fine‐Tuned Heme Biosynthesis

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