Role of the Iron-Sulfur Cluster in Human Ferrochelatase in Sensing Changes in Mitochondrial Physiology

Elinor Stanley Author

04/02/2021 Added

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Investigating the role of FECH in sensing changes in mitochondrial physiology with regards to redox potential and pH sensing

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[00:00:00.840]Good day everyone.
[00:00:01.800]My name is Elinor Stanley and my presentation today is over the role of the
[00:00:05.280]iron sulfur cluster in human ferrochelatase
[00:00:07.450]in sensing changes in mitochondrial physiology.
[00:00:10.590]This project was sponsored by the UNL UCARE program and Dr. Oleh Khalimonchuk,
[00:00:14.270]while experiment and research process was aided by Jonathan Dietz. To
[00:00:18.880]begin, we will start with background information for this project.
[00:00:21.510]Heme is a crucial cofactor synthesized by conservative pathway in the
[00:00:24.960]mitochondria it's synthesis begins in the matrix of the mitochondria continues
[00:00:29.130]in the cytosol,
[00:00:30.000]then returns to the matrix of the mitochondria where ferrochelatase inserts
[00:00:33.420]iron in a protoporphyrin nine to produce heme.
[00:00:36.540]Ferrochelatase is a dimeric protein,
[00:00:38.710]found on the matrix side of the mitochondrial inner membrane.
[00:00:41.280]The product of ferrochelatase, heme, is required in several cellular
[00:00:45.330]functions, including electron transport, gas sensing, signal transduction,
[00:00:50.100]iron homeostasis, and xenobiotic detoxification.
[00:00:53.640]There are many clinical manifestations resulting from heme homeostasis
[00:00:57.030]disruptions. These include anemia, porphyria,
[00:01:01.470]cardiovascular disorders,
[00:01:02.820]and neurodegenerative syndromes. in order to maintain proper heme homeostasis to
[00:01:07.500]prevent some of these disorders,
[00:01:09.030]mitochondrial function and physiology must be maintained.
[00:01:12.420]Previous studies have shown that mitochondrial pH and redox potential affect
[00:01:16.290]heme biosynthesis. In zebra fish,
[00:01:18.570]the deletion of the enzyme ATPase inhibitory factor one,
[00:01:22.170]or ATPIF1 causes mitochondrial pH to increase while
[00:01:26.400]mitochondrial redox potential decreases. The zebrafish lacking in ATPF1
[00:01:31.060]also presented with symptoms relating to anemia. upon further investigation,
[00:01:35.910]It was found that in these animals, less heme is synthesized and ferrochelatase
[00:01:39.870]enzymatic activity is significantly decreased.
[00:01:42.900]Both of these phenotypes would contribute to the animal's anemia.
[00:01:46.350]Zebrafish, and human FECH,
[00:01:48.870]contain a two iron two sulfur cluster,
[00:01:53.130]but its role in FECH is unclear. these phenomena along with the susceptibility of
[00:01:57.300]iron sulfur clusters
[00:01:58.380]to changes in redox potential point to the two iron to sulfur cluster in FECH
[00:02:03.240]being a sensor to changes in mitochondrial redox potential. However,
[00:02:06.750]this hypothesis requires further investigation.
[00:02:10.620]The purpose of this study is to determine the role of the iron sulfur cluster in
[00:02:14.220]human ferrochelatase using the yeast Saccharomyces cerevisiae genetic model.
[00:02:19.650]We have decided to use Saccharomyces cerevisiae because it contains its own
[00:02:23.520]homologue effect known as hem15,
[00:02:26.130]but this enzyme does not include a two iron two sulfur cluster,
[00:02:29.370]and we were able to express human genes in this organism to study their
[00:02:32.400]function.
[00:02:33.480]The specific aims of the study were to develop a yeast strain with endogenous
[00:02:37.350]alterations to mitochondrial pH and redox potential,
[00:02:40.950]as well as analyze the pH and redox potential sensing of the iron sulfur cluster
[00:02:45.090]of human FECH.
[00:02:46.920]First one to determine if human FECH was functional in yeast,
[00:02:50.460]so expressed human FECH in a yeast strain lacking hem15.
[00:02:54.270]This strain is unable to grow without heme supplementation,
[00:02:57.000]so we can easily assess the functionality of FECH in the system. To analyze this,
[00:03:01.570]we did a growth spot assay on media containing and lacking heme. Human
[00:03:06.280]FECH was stably expressed and functional in the yeast strain lacking the yeast
[00:03:10.180]enzyme also known as hem15
[00:03:11.740]knockout, as shown by the glucose growth without heme supplementation.
[00:03:16.570]We're also able to confirm its stable expression via Western blot.
[00:03:20.650]Interestingly,
[00:03:21.640]when the two iron two sulfur cluster in human FECH is disrupted by introducing a
[00:03:26.080]serine mutation at the cystine-406,
[00:03:29.680]FECH is no longer able to complement the hem15 deficiency,
[00:03:33.220]meaning that is unable to produce heme for the cell to survive. From this,
[00:03:36.970]we can conclude that the two iron two sulfur cluster in human FECH is required
[00:03:40.630]for proper FECH function.
[00:03:42.910]We next assessed if both yeast and human FECH assembled into high molecular
[00:03:47.230]weight complexes similarly.
[00:03:48.220]We used high velocity sucrose gradient fractionation to
[00:03:52.840]determine this.
[00:03:54.970]This assay separates protein complexes by molecular weight,
[00:03:58.900]by introducing them to a 10% to 50% sucrose gradient solution.
[00:04:04.030]Once mitochondria are loaded into the sucrose gradient solution,
[00:04:06.820]they are subjected to centrifugation, where smaller protein complexes migrate
[00:04:10.570]towards the lower sucrose concentration and larger protein complexes migrate
[00:04:14.680]towards the higher sucrose concentration. Using high velocity
[00:04:17.950]sucrose gradient fractionation,
[00:04:20.230]we found that yeast hem15 and human FECH were found to assemble in
[00:04:24.910]250 to 400 kDa and 150 to 350 kDa
[00:04:29.920]high-mass complexes respectively. For future work,
[00:04:33.520]we are going to generate the ATPF1 or INH1 (the yeast
[00:04:38.440]homologue) knockouts in wild-type and hem15 deficient yeast, also known as INH1
[00:04:43.300]knockout and hem15 INH1 double knockout,
[00:04:47.560]Next we're
[00:04:48.550]going to express plasmids bearing the hem15, the FECH
[00:04:53.320]C406S and FECH in the hem15 INH1 double knockout. We'll then perform
[00:04:58.270]a growth spot essay for the transformants under heme replete
[00:05:01.450]and deplete conditions, and isolate the mitochondria from those transformants
[00:05:06.460]Using those transformants will analyze the steady state levels of hem15, FECH
[00:05:10.930]and FECH C406S in mitochondria,
[00:05:13.810]in the absence of INH1. We'll monitor the ferrochelatase activity of hem15
[00:05:18.820]FECH and FECH C406S in the absence of INH1
[00:05:23.230]And finally we'll determine the high molecular weight assembly of hem15, FECH
[00:05:27.220]and FECH C406S in the absence of INH1. However,
[00:05:31.630]we hypothesize that each of these ferrochelatase variants will respond to the
[00:05:35.380]absence of INH1 differently. Since Hem15
[00:05:39.010]lacks the iron sulfur cluster.
[00:05:40.810]We expect it to be unaffected by the changes in redox potential and the INH1
[00:05:44.710]knockout. However, for human FECH,
[00:05:47.440]we expect it to be unable to function in response to the drop in redox
[00:05:50.590]potential.
[00:05:51.160]When INH1 is deleted in similar fashion to zebrafish,
[00:05:56.260]it is likely that FECH C406S will not act differently when INH! is
[00:06:00.920]absent.
[00:06:01.700]I also predict that the high molecular weight assembly of hem15 and FECH will be
[00:06:05.300]the same as under wild type conditions.
[00:06:08.390]Thank you so much for your time and attention, have a great day and go big red!

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Role of the Iron-Sulfur Cluster in Human Ferrochelatase in Sensing Changes in Mitochondrial Physiology

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