Monitoring iron-sulfur cluster occupancy across the E. coli proteome using chemoproteomics

Iron-sulfur (Fe-S) clusters are ubiquitous metallocofactors found across diverse protein families, where they perform myriad functions including redox chemistry, radical generation, and gene regulation. Monitoring Fe-S cluster occupancy in protein targets directly within native biological systems has been challenging. Commonly utilized spectroscopic methods to detect Fe-S clusters require purification of proteins prior to analysis. Global iron incorporation into the proteome can be monitored using radiolabeled iron, but limitations include the low resolution afforded by gel-based autoradiography. Here, we report the development of a mass spectrometry-based strategy to assess Fe-S cluster binding in a native proteome. This chemoproteomic strategy relies on monitoring changes in the reactivity of Fe-S cluster cysteine ligands upon disruption of Fe-S cluster incorporation. Application to E. coli cells cultured under iron-depleted conditions enabled monitoring of disruptions to Fe-S cluster incorporation broadly across the E. coli Fe-S proteome. Evaluation of E. coli deletion strains of three scaffold proteins within the Isc Fe-S biogenesis pathway enabled the identification of Fe-S clients that are reliant on each individual scaffold protein for proper cluster installation. Lastly, cysteine-reactivity changes characteristic of Fe-S ligands were used to identify previously unannotated Fe-S proteins, including the tRNA hydroxylase, TrhP, and a member of a family of membrane transporter ATPase subunits, DppD. In summary, the chemoproteomic strategy described herein provides a powerful tool to report on Fe-S cluster incorporation directly within a native proteome, to interrogate the role of scaffold and accessory proteins within Fe-S biogenesis pathways, and to identify previously uncharacterized Fe-S proteins.

Iron incorporation both intra- and extra-cellularly improves the yield and saccharification of switchgrass (Panicum virgatum L.) biomass

Background Pretreatments are commonly used to facilitate the deconstruction of lignocellulosic biomass to its component sugars and aromatics. Previously, we showed that iron ions can be used as co-catalysts to reduce the severity of dilute acid pretreatment of biomass. Transgenic iron-accumulating Arabidopsis and rice plants exhibited higher iron content in grains, increased biomass yield, and importantly, enhanced sugar release from the biomass. Results In this study, we used intracellular ferritin (FerIN) alone and in combination with an improved version of cell wall-bound carbohydrate-binding module fused iron-binding peptide (IBPex) specifically targeting switchgrass, a bioenergy crop species. The FerIN switchgrass improved by 15% in height and 65% in yield, whereas the FerIN/IBPex transgenics showed enhancement up to 30% in height and 115% in yield. The FerIN and FerIN/IBPex switchgrass had 27% and 51% higher in planta iron accumulation than the empty vector (EV) control, respectively, under normal growth conditions. Improved pretreatability was observed in FerIN switchgrass (~ 14% more glucose release than the EV), and the FerIN/IBPex plants showed further enhancement in glucose release up to 24%. Conclusions We conclude that this iron-accumulating strategy can be transferred from model plants and applied to bioenergy crops, such as switchgrass. The intra- and extra-cellular iron incorporation approach improves biomass pretreatability and digestibility, providing upgraded feedstocks for the production of biofuels and bioproducts.

Use of Ppmx-T003 As a Potent Inhibitor of Erythrocytosis in Polycythemia Vera

Iron incorporation through transferrin (Tf) and its receptor, TfR1, plays a critical role in erythropoiesis. We hypothesized that blockade of TfR1 may be a promising strategy to suppress erythrocytosis observed in polycythemia vera (PV). To examine this possibility, we developed PPMX-T003, a monoclonal antibody against human TfR1, through phage display screening. PPMX-T003 was observed to bind to TfR1 with a high affinity(Kd= 3.56×10-10)and presented strong ability to suppress the growth of PV model cell lines such as HEL (EC50= 0.07 nM). PPMX-T003 induced TfR1 internalization but blocked the recycling of the receptor presumably because it was strongly bound, even in the endosome, which switched the fate of TfR1 from recycling to degradation. Consequently, the PPMX-T003 treatment greatly reduced the intracellular iron concentration, along withcell cycle arrest in the G2/M phase in HEL cells. In contrast, PPMX-T003 failed to block cell proliferation in human umbilical vein endothelial cells that expressed a subtle level of TfR1. These findings suggest that PPMX-T003 is a potent inhibitor for cells whose growth is dependent on iron incorporation by TfR1. This led us to examine whether PPMX-T003 blocked the endogenous erythroid colony (EEC) formation in erythroid progenitor cells in PV. We observed that PPMX-T003, at a concentration of 40 ng/mL, exhibited complete blockade of EEC formation inJAK2 V617F-positive PV patients' cells. Although hydroxycarbamide (HU) and phlebotomy are well-established treatment strategies for the management of hematocrit in PV patients, there is a risk of secondary leukemia associated with HU treatment and side effects such as fatigue and restless leg syndrome due to phlebotomy-induced iron deficiency. Therefore, we suggest PPMX-T003, which may preferentially suppress erythrocytosis without changing the iron level in peripheral blood, as an alternative treatment for PV patients. Disclosures Ohira: PPMX: Current Employment, Current equity holder in private company. Nomura:PPMX: Current Employment. Matsuura:PPMX: Current Employment. Komatsu:Takeda Pharmaceutical Co., Ltd, Novartis Pharma KK, Shire Japan KK: Speakers Bureau; AbbVie: Other: member of safety assessment committee in M13-834 clinical trial.; PPMX: Consultancy, Research Funding; Meiji Seika Pharma Co., Ltd.: Patents & Royalties: PCT/JP2020/008434, Research Funding; Otsuka Pharmaceutical Co., Ltd., PharmaEssentia Japan KK, AbbVie GK, Celgene KK, Novartis Pharma KK, Shire Japan KK, Japan Tobacco Inc: Consultancy; Otsuka Pharmaceutical Co., Ltd., Shire Japan KK, Novartis Pharma KK, PharmaEssentia Japan KK, Fuso Pharmaceutical Industries, Ltd., Fujifilm Wako Pure Chemical Corporation, Chugai Pharmaceutical Co., Ltd., Kyowa Hakko Kirin Co., Ltd., Takeda Pharmaceutica: Research Funding.

Disclosing the Molecular Mechanism of Iron Incorporation in Listeria innocua Dps by EPR Spectroscopy

Bacteria overexpress, under condition of starvation or oxidative stress, Dps (DNA-binding proteins from starved cells), hollow sphere formed by 12 identical subunits endowed with ferritin-like activity. The iron oxidation and incorporation in Dps take place using H2O2 produced under starvation as preferred iron oxidant, thereby protecting bacteria from oxidative damage. Even if the role of Dps is well known, the mechanism of iron oxidation and incorporation remain to be elucidated. Here, we have used the EPR technique to shed light on the Fe(II) binding and oxidation mechanism at the ferroxidase center using both the wild-type (wt) protein and mutants of the iron ligands (H31G, H43G and H31G-H43G-D58A). The EPR titration of wt Dps and the H31G mutant with Fe(II) upon H2O2 addition shows that Fe(II) is oxidized with the increase of the signal at g = 4.3, reaching a maximum for 12 Fe(II)/subunit. The EPR signal becomes negligible when the titration is carried out on the triple mutant. These experiments indicate that the iron firstly occupied the A site at the ferroxidase center and confirm that the residues H31, H43 and D58 have a key role in the iron oxidation and incorporation process. Moreover, the data indicate that the ferroxidase center, upon mutation of H31 or H43 to Gly, changes the mode of iron binding. Finally, we demonstrate here that, when the iron micelle forms, the EPR signal at g = 4.3 disappears indicating that iron leaves the ferroxidase center to reach the inner cavity.