iron sulphur cluster assembly

[ValWood]

Antonias old notes!
I'm very good at "tidying away" (chucking) old notes so thought I'd upload this here for ref

Originally posted by @Antonialock in pombase/curation#1032 (comment)

[hattrill]

Fly iron sulphur cluster assembly, from PMID:29491838

GO: mitochondrion + iron-sulfur cluster assembly	In diagram
bcn92	y
fh	y
Nfs1	y
IscU	y
Nubpl	n
Hsc70-5	y
Fdx1	y
MagR (IscA1)	y
ABCB7	y
Fdx2	y
CG8043 (Iba57)	y
CG13623 (IscA2)	y
CG31126 (BolA1)	y
CG32857 (Nfu)	y
CG33502 (Nfu)	y
Mocs1	y
Hsc20	y
CG32500 (Nfu)	y

Nubpl is not on the diagram. It is an ATP-dependent FeS cluster chaperone involved in the assembly of Complex I.

In diagram but no annotation to iron-sulfur cluster assembly
dare
Grx5
CG33672 ( ortholog of BOLA2/BOLA2B)

[ValWood]

@PCarme has curated our pathway
http://noctua.geneontology.org/workbench/noctua-visual-pathway-editor/?model_id=gomodel%3A6690711d00000331
so we will align orthologs into the table

[hattrill]

The definition of iron-sulfur cluster assembly GO:0016226 is quite narrow:
"The incorporation of iron and exogenous sulfur into a metallo-sulfur cluster."
and I am wondering where the end/edges are so would be good to compare with your Pombe model

[hattrill]

Fly mitochondrial iron-sulfur cluster assembly (Dmel) gomodel:6796b94c00000657

Has components:

bcn92 Dmel [2Fe-2S] cluster assembly
fh Dmel [2Fe-2S] cluster assembly
Grx5 Dmel [2Fe-2S] cluster assembly
IscU Dmel [2Fe-2S] cluster assembly
IscU Dmel [2Fe-2S] cluster assembly
ND-ACP Dmel [2Fe-2S] cluster assembly
Nfs1 Dmel [2Fe-2S] cluster assembly
CG8043 Dmel [4Fe-4S] cluster assembly
dare Dmel iron-sulfur cluster assembly
Fdx1 Dmel iron-sulfur cluster assembly
Hsc20 Dmel [2Fe-2S] cluster assembly
Hsc70-5 Dmel [2Fe-2S] cluster assembly

Upstream or downstream
mfrn Dmel iron import into the mitochondrion
Nubpl Dmel mitochondrial respiratory chain complex I assembly
CG31126 Dmel protein maturation
CG32500* Dmel protein maturation
ABCB7 Dmel iron-sulfur cluster export from the mitochondrion

*CG32500, CG33502, CG32857 are all orthologs of NFU. Only modelled CG32500

Made some notes to accompany this pathway as there are some differences and some areas of the biology are a bit fuzzy:

Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-Sulfur (Fe-S) assembly pathway involves the incorporation of iron and exogenous sulfur into organometallic Fe-S clusters. Iron-sulfur clusters are incorporated into various proteins and are required as co-factors for a variety of processes such as electron transfer, catalysis and structural roles. Fe-S assembly pathway starts in the mitochondria PMID:29491838. The mitochondrial Fe-S Fe-S assembly pathway is sometimes referred to as the Iron-Sulfur Cluster (ICS) System and between prokaryotes to eukaryotes.
The Mitoferrin (Dmel mfrn) transporter mediates mitochondrial Fe2+ uptake PMID: 29491838 [MF: ferrous iron transmembrane transporter activity, BP: iron import into the mitochondrion, CC: mitochondrial inner membrane]. Iron-sulfur cluster assembly enzyme (Dmel IscU) is a scaffold protein on which [2Fe-2S] clusters are assembled. IscU [MF:ferrous iron binding, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix]. NFS1 L-cysteine desulfurase (Dmel Nfs1) provides the inorganic sulfide as persulfide. NFS1 catalyzes the removal of elemental sulfur from cysteine to produce alanine (PMID:29491838) (MF: cysteine desulfurase activity, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix]. The ISC complex is responsible for cluster assembly and contains NFS), ISD1/LYRM4( Dmel bcn92), and acyl carrier protein (Acp, NDUFAB1, Dmel ND-ACP). NDUFAB1/ACP is also a subunit for Mitochondrial Complex I Accessory Subunit and this appears to be a ‘moonlighting’ function. Structural studies of the NFS1–ISD11–ACP (SDA) subcomplex show that a 4′-phosphopantetheine–conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, which stabilizes the complex. Steady-state kinetic analysis on SDA revealed a significantly lower KM for l-cysteine in comparison with the previously characterized complex that lacked ACP (PMID:28634302). The activity of bcn92 and ND-ACP have therefore been modeled as MF:structural molecule activity ( BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix ‘directly positively regulates’ Nfs1; MF: cysteine desulfurase activity]. Frataxin, FXN, (Dmel fh) acts as an allosteric activator (PMID:286343020) [enzyme activator activity, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix], stimulating transfer of the NFS1-bound persulfide to ISCU which has Fe2+ bound Nfs1 MF:sulfur carrier activity, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix].ISCU-persulfide is reduced to sulfide via an FDX2-dependent electron transfer (Dmel Fdx1 is orthologous to mammalian FDX2) (Note:In vivo, FDX2 is characterised as the ferrodoxin that mediates this.In vitro both FDX1 and FDX2 interact with the mitochondrial [2Fe-2S] assembly complex ISC) protein complex. FDX2 binds more tightly to the cysteine desulfurase complex than FDX1 does and the rate of cluster assembly was faster with FDX2 than with FDX1 PMID:28001042.) The [2Fe-2S] cluster is formed by interaction of two [1Fe-1S]-containing ISCU on different core ISC complexes [IscU: MF:iron-sulfur cluster chaperone activity, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix], Fdx1 ‘provides_input for’ this via [MF:electron transfer activity; iron-sulfur cluster chaperone activity, BP:iron-sulfur cluster assembly, CC:mitochondrial matrix]. Dmel Dare is the NADPH-ferrodoxin reductase for Fdx1.

Iron–sulfur cluster biogenesis in mammalian cells: New insights into the molecular mechanisms of cluster delivery

The newly made [2Fe-2S]2+ is then released from ISCU and transferred to the monothiol GLRX5 (Dmel Grx5; 2 iron, 2 sulfur cluster binding) with the help of the HSPA9/HSC20 (Dmel Hsc70-5/ Hsc20: MF:ATP-dependent protein folding chaperone/ATPase activator activity, BP:[2Fe-2S] cluster assembly, CC:mitochondrial matrix) chaperone pair (PMID:25245479). “Cluster transfer from GLRX5 and insertion into [2Fe-2S] target apoproteins occurs spontaneously. Dislocation of the GLRX5-bound [2Fe-2S] cluster, fusion to a [4Fe-4S] cluster and its insertion into thiol-reduced apoproteins requires ISCA1–ISCA2, IBA57 (Dmel MagR-CG13623, CG8043, as well as the electron transfer chain NADPH, ferredoxin reductase FDXR (Dmel Dare) , and ferredoxin FDX2 (Dmel Fdx1). The latter system catalyzes the reductive fusion of [2Fe-2S]2+ to [4Fe-4S]2+ clusters in a IBA57-dependent fashion. Despite its structural similarity, the FDX2 paralog FDX1 is not functional in this reaction.” (PMID:32817474). Modelled this as mitochondrial [4Fe-4S] assembly complex (has_part MagR, CG13623) [MF:2 iron, 2 sulfur cluster binding,
BP:[4Fe-4S] cluster assembly, CC:mitochondrial matrix] directly positively regulated by CG8043 [MF:molecular_function, BP:[4Fe-4S] cluster assembly, CC:mitochondrial matrix]. Fdx1 ‘provides_input for’ this via [MF:electron transfer activity; iron-sulfur cluster chaperone activity, BP:iron-sulfur cluster assembly, CC:mitochondrial matrix]. Dmel Dare is the NADPH-ferrodoxin reductase for Fdx1. The result of this is modelled as mitochondrial [4Fe-4S] assembly complex (has_part MagR, CG13623) [MF:4 iron, 4 sulfur cluster binding,
BP:[4Fe-4S] cluster assembly, CC:mitochondrial matrix]
,

Downstream from Grx5 and mitochondrial [4Fe-4S] assembly complex [2Fe2S] and [4Fe4S], respectively, are transferred to other proteins.

Grx5 provides [2Fe2S] clusters for export by ABCB7, (Dmel ABCB7) but probably via some uncharacterised intermediated so modeled as causally upstream of, positive effect [MF:ABC-type iron-sulfur cluster transporter activity, BP:iron-sulfur cluster export from the mitochondrion, CC: mitochondrial inner membrane] or transfer to apo-proteins to make [2Fe2S]-containing proteins.

The mitochondrial [4Fe-4S] assembly complex provides [4Fe-4S] for NFU1 (Dmel CG32500). [ MF:4 iron, 4 sulfur cluster binding, BP:protein maturation*, CC:mitochondrial matrix]

*not sure where ‘iron-sulfur cluster assembly’ should end and protein maturation should begin as iron-sulfur cluster assembly defined as just the ‘The incorporation of iron and exogenous sulfur into a metallo-sulfur cluster.’ and does not include the insertion of a prosthetic group.

Downstream of CG32500:

IND1/NUBPL (Dmel Nubpl) which is required for mitochondrial Complex I assembly [MF:ATP-dependent FeS chaperone activity, BP:mitochondrial respiratory chain complex I assembly, CC:mitochondrial inner membrane]

CG31126 is the ortholog of BolA1 (no close ortholog for BolA3). Might have some role in the biogenesis of other [4Fe-4S]-containing complexes. Modeled as [MF:molecular_function, BP:protein maturation, CC:mitochondrial matrix] as role still unclear.

Or other [4Fe-4S]-containing proteins

[ValWood]

Our pathways currently look quite different, so using the combined information, it might be possible to extend ours or even both.

As a first step, @PCarme could you add the pombe entities as a column to the table above (and maybe the human symbols at the same time, since they will likely more closely align with some of the pathways diagrams) for future reference.
Then perhaps we should use complexes in the same places as @hattrill now we know how!
Also add the main inputs and outputs.
This will also allow us to more easily check that we use the same relationships in the same places, and have covered the same entities.
cheers! no hurry.

[hattrill]

I've made some notes on the differences, so if you want to have a chat about it any time.....there are some difficulties deciding what MFs are appropriate and where to end the pathway so would be good to converge. I didn't add in the GO CC for the 'mitochondrial [2Fe-2S] assembly complex' as I wanted to check with Kimberly et al about whether how to do this when you know the MF for the individual SUs for this.

These are the SUs of the 'mitochondrial [2Fe-2S] assembly complex':

FBgn0011361 ND-ACP
FBgn0037637 IscU
FBgn0013432 bcn92
FBgn0030092 fh
FBgn0032393 Nfs1

I have a table of the Pombe orthologs, so I will add that later - just need to add the human ones.

[ValWood]

You can get most of the human ones quickly by adding the pombe IDs here:
https://www.pombase.org/query/start_from/gene_list
and then click on the list number.
Click the [download] button to the left.
Select human orthologs
(if you don't already have them)

[hattrill]

Fly	Pombe	Human
bcn92	isd11	ISD11
fh	fxn1	FXN
Grx5	grx5	GLRX5
IscU	isu1	ISCU
ND-ACP	SPAC4H3.09	NDUFAB1
Nfs1	nfs1	NFS1
CG8043	iba57	IBA57
dare	arh1	FDXR
Fdx1	etp1	FDX2
Hsc20	jac1	HSCB
Hsc70-5	ssc1	HSPA9

Upstream or Downstream

Fly	Pombe	Human
mfrn	mrs3	SLC25A28/SLC25A37
Nubpl	None	NUBPL
CG31126	uvi31	BOLA1
None	fra3	BOLA3
CG32500/CG33502/CG32857	nfu1	NFU1
ABCB7	atm1	ABCB7

[ValWood]

pretty well conserved then!

[PCarme]

Thanks @hattrill , I was also filling the comparative table. I have added the genes that are annotated to "mitochondrion" and "iron-sulfur cluster assembly" in PomBase that were not in the previous table as well.

Fly	Pombe	Human
bcn92	isd11	LYRM4
fh	fxn1	FXN
Nfs1	nfs1	NFS1
IscU	isu1	ISCU
Nubpl		NUBPL
Hsc70-5	ssc1	HSPA9
Fdx1	etp1	FDX2
MagR (IscA1)	isa1	ISCA1
ABCB7	atm1	ABCB7
Fdx2		FDX1
CG8043 (Iba57)	iba57	IBA57
CG13623 (IscA2)	isa2	ISCA2
CG31126 (BolA1)	uvi31	BOLA1
CG32857 (Nfu)	nfu1	NFU1
CG33502 (Nfu)	nfu1	NFU1
Mocs1		MOCS1
Hsc20	jac1	HSCB
CG32500 (Nfu)	nfu1	NFU1
CIAPIN1	dre2	CIAPIN1
CG13667	tah18	NDOR1
Roe1	mge1	GRPEL1, GRPEL2
Grx5	grx5	GLRX5
ND-ACP	SPAC4H3.09	NDUFAB1

I noticed that uvi31 isn't annotated to any process term at the moment, I think we could use an ISO to the cerevisiae ortholog BOL1.

Note that our pathway model is not only the mitochondrial iron-sulfur cluster assembly, it also integrates the mitochondrial export and cytosolic iron-sulfur cluster assembly.
I'll compare my model with the fly one to see what could be added to our pathway

[hattrill]

Cytosolic bit still to do for me - I shall have a look at yours.

[hattrill]

Looking at the cytosolic portion, think we need a new MF for the:
tah18 -> dre2 (Spombe)
CG13667 -> CIAPIN1 (Dmel)
NDOR1 -> CIAPIN1 (Human)
step

crresponding to https://www.rhea-db.org/rhea/67716

Prob something like:
NADPH-iron-sulfur [2Fe-2S] protein oxidoreductase activity
Catalysis of the reaction: 2 oxidized [2Fe-2S]-[protein] + NADPH = 2 reduced [2Fe-2S]-[protein] + NADP+ + H+

[PCarme]

Our pathways currently look quite different, so using the combined information, it might be possible to extend ours or even both.

As a first step, @PCarme could you add the pombe entities as a column to the table above (and maybe the human symbols at the same time, since they will likely more closely align with some of the pathways diagrams) for future reference. Then perhaps we should use complexes in the same places as @hattrill now we know how! Also add the main inputs and outputs. This will also allow us to more easily check that we use the same relationships in the same places, and have covered the same entities. cheers! no hurry.

Here is the updated version of our model http://noctua.geneontology.org/workbench/noctua-alliance-pathway-preview/?model_id=gomodel%3A6690711d00000331
I have added the main inputs/outputs of the pathway, and changed a couple of functional links I had made based on previous assumptions that more recent reviews and studies don't really support (like fxn1 providing iron to isu1 for example).
I have also displayed the complexes for which the individual activities of the different subunits are unclear as complexes in the model, so it looks much more like the fly model now (at least for the mitochondrial part of the pathway).

[hattrill]

Done the cytosolic portion - pretty much same as yours.
Have asked for term for NADPH-iron-sulfur [2Fe-2S] protein oxidoreductase activity
geneontology/go-ontology#29668

[hattrill]

adding cytosolic genes and orthologs for completeness:

Human	Dmel	Pombe	sc
NUBP2	Nubp2	SPAC806.02c	CFD1
NUBP1	Nubp1	nbp35	NBP35
CIAPIN1	CIAPIN1	dre2	DRE2
NDOR1	CG13667	tah18	TAH18
CIAO3	CG17683	nar1	NAR1
CIAO1	Ciao1	SPAC806.02c	CIA1
CIAO2B	galla-2	cia2	CIA2
MMS19	Mms19	mms19	MET18
CIAO2A	galla-1	none	none*

*Did not model galla-1 for now as seems to be context-dependent.