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

Elinor Stanley Author

04/02/2021 Added

76 Plays

Description

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

Searchable Transcript

Search:

[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!

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

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

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments