Fe-S Cluster-containing Proteins Research Articles

THU083 Disulfiram Mediated Anti-tumor Effect In Pituitary Neuroendocrine Tumors By Inducing Cuproptosis

Abstract Disclosure: N. Huang: None. Y. Zhang: None. Y. Liu: None. Y. Feng: None. L. Xue: None. Z. Wu: None. Pituitary neuroendocrine tumors (PitNETs) are common intracranial tumors. Dopamine agonists and somatostatin analogues are the first-line drugs for PRL and GH-secreting PitNETs clinically. However, there is no effective drug for other subtypes and drug-resistant PitNETs. Thus, it’s impending to discover new therapeutic targets and medicine. Cuproptosis is a copper-dependent form of cell death driven by lipoylated protein aggregation, loss of Fe-S cluster-containing proteins, and induction of HSP70. However, the role of cuproptosis in PitNETs is not clear. In this study, a total of 17 primary samples of PitNETs were collected to perform high-throughput drug screening, an anti-abuse drug disulfiram (DSF) was identified that can induce cell death effectively. We found that disulfiram also has a strong inhibitory effect on pituitary tumor cell lines MMQ, Att20 and GH3 (-87.1%, -62.5%, -92.7%, p<0.001), but the inhibitory effect was eliminated by copper chelator TTM/BCS. This indicated that DSF played the anti-tumor effect requiring the participation of copper ions. To explore the underlying mechanism that DSF induces the cell death of PitNETs, we detected the hallmark of cuproptosis and found that DSF leads to a decrease in the acylation level of proteins related to the tricarboxylic acid cycle, limiting the function of the protein. On the one hand, DSF leads to the downregulation of iron-sulfur cluster-associated proteins, and the oligomerization of dihydrolipoamide S-acetyltransferase (DLAT), which is eventually accompanied by an increase in the protein level of heat shock protein 70 (HSP70) at protein and mRNA level, causing proteotoxic stress leading to cell death. Finally, DSF treatment inhibited the tumor growth in MMQ cell transplanted mouse model and destructed the morphology of the pituitary tumor 3D sphere similarly demonstrating the inhibition effect. In conclusion, we find that the anti-abuse drug disulfiram can induce coproptosis in pituitary tumor cells in a copper-dependent manner and is a potential drug for the treatment of PitNETs. Presentation: Thursday, June 15, 2023

Interaction of client-the scaffold on which FeS clusters are build-with J-domain protein Hsc20 and its evolving Hsp70 partners.

In cells molecular chaperone systems consisting of Hsp70 and its obligatory J-domain protein (JDP) co-chaperones transiently interact with a myriad of client proteins—with JDPs typically recruiting their partner Hsp70 to interact with particular clients. The fundamentals of this cyclical interactions between JDP/Hsp70 systems and clients are well established. Much less is known about other aspects of JDP/Hsp70 system function, including how such systems evolved over time. Here we discuss the JDP/Hsp70 system involved in the biogenesis of iron-sulfur (FeS) clusters. Interaction between the client protein, the scaffold on which clusters are built, and its specialized JDP Hsc20 has stayed constant. However, the system’s Hsp70 has changed at least twice. In some species Hsc20’s Hsp70 partner interacts only with the scaffold, in others it has many JDP partners in addition to Hsc20 and interacts with many client proteins. Analysis of this switching of Hsp70 partners has provided insight into the insulation of JDP/Hsp70 systems from one another that can occur when more than one Hsp70 is present in a cellular compartment, as well as how competition among JDPs is balanced when an Hsp70 partner is shared amongst a number of JDPs. Of particularly broad relevance, even though the scaffold’s interactions with Hsc20 and Hsp70 are functionally critical for the biogenesis of FeS cluster-containing proteins, it is the modulation of the Hsc20-Hsp70 interaction per se that allows Hsc20 to function with such different Hsp70 partners.

Characterization of Fe-S Clusters in Proteins by Mӧssbauer Spectroscopy.

57Fe Mӧssbauer spectroscopy is unparalleled in the study of Fe-S cluster-containing proteins because of its unique ability to detect all forms of iron. Enrichment of biological samples with the 57Fe isotope and manipulation of experimental parameters such as temperature and magnetic field allow for elucidation of the number of Fe-S clusters present in a given protein, their nuclearity, oxidation state, geometry, and ligation environment, as well as any transient states relevant to enzyme chemistry. This chapter is arranged in five sections to help navigate an experimentalist to utilize 57Fe Mӧssbauer spectroscopy for delineating the role and structure of biological Fe-S clusters. The first section lays out the tools and technical considerations for the preparation of 57Fe-labeled samples. The choice of experimental parameters and their effects on the Mӧssbauer spectra are presented in the following two sections. The last two sections provide a theoretical and practical guide on spectral acquisition and analysis relevant to Fe-S centers.

The ErpA/NfuA complex builds an oxidation-resistant Fe-S cluster delivery pathway

Fe-S cluster-containing proteins occur in most organisms, wherein they assist in myriad processes from metabolism to DNA repair via gene expression and bioenergetic processes. Here, we used both in vitro and in vivo methods to investigate the capacity of the four Fe-S carriers, NfuA, SufA, ErpA, and IscA, to fulfill their targeting role under oxidative stress. Likewise, Fe-S clusters exhibited varying half-lives, depending on the carriers they were bound to; an NfuA-bound Fe-S cluster was more stable (t½ = 100 min) than those bound to SufA (t½ = 55 min), ErpA (t½ = 54 min), or IscA (t½ = 45 min). Surprisingly, the presence of NfuA further enhanced stability of the ErpA-bound cluster to t½ = 90 min. Using genetic and plasmon surface resonance analyses, we showed that NfuA and ErpA interacted directly with client proteins, whereas IscA or SufA did not. Moreover, NfuA and ErpA interacted with one another. Given all of these observations, we propose an architecture of the Fe-S delivery network in which ErpA is the last factor that delivers cluster directly to most if not all client proteins. NfuA is proposed to assist ErpA under severely unfavorable conditions. A comparison with the strategy employed in yeast and eukaryotes is discussed.

Structural Characterization of Poised States in the Oxygen Sensitive Hydrogenases and Nitrogenases.

The crystallization of FeS cluster-containing proteins has been challenging due to their oxygen sensitivity, and yet these enzymes are involved in many critical catalytic reactions. The last few years have seen a wealth of innovative experiments designed to elucidate not just structural but mechanistic insights into FeS cluster enzymes. Here, we focus on the crystallization of hydrogenases, which catalyze the reversible reduction of protons to hydrogen, and nitrogenases, which reduce dinitrogen to ammonia. A specific focus is given to the different experimental parameters and strategies that are used to trap distinct enzyme states, specifically, oxidants, reductants, and gas treatments. Other themes presented here include the recent use of Cryo-EM, and how coupling various spectroscopies to crystallization is opening up new approaches for structural and mechanistic analysis.

Cracking Open a Molecular Calculator: DNA Charge Transport and Primase

De novo initiation of DNA synthesis by DNA polymerases requires formation of an initial “primer” on the template. In eukaryotes, this function is provided by a distinct DNA primase. Primase, a DNA-dependent RNA polymerase, generates short (8-12 nt) RNA primers on ssDNA templates. These initial RNA primers are handed off to DNA polymerase α (pol α), which extends the primer by ∼20 nts. This primed RNA/DNA substrate is then handed off to processive polymerases e or δ, which synthesize the vast majority of new DNA on the leading and lagging strands. X-ray crystal structures of human primase have been used to propose models for the catalytic mechanism. However, the molecular basis for primer counting and handoff remains unclear. Primase contains a 4Fe-4S cluster in the p58C domain critical to pol-prim loading and activity, but its specific function is not known. We are investigating the hypothesis that the function of the cluster is to drive primer length counting. Well-stacked duplex DNA has been shown capable of transporting charge over long distances, and it has been proposed this phenomenon enables communication between Fe-S cluster-containing proteins. In this study, we show the 4Fe-4S cluster in wild-type p58C is capable of mediating charge transport (CT) with DNA. DNA-modified electrodes were used to characterize the electrochemistry of p58C. Based on previously determined WT structures, we identified a possible pathway from the cluster to the DNA binding site in p58C and designed mutants of key residues in this pathway. Mutation of Tyr345 to Phe greatly diminishes DNA CT. Primase activity assays with WT and mutant proteins revealed substantial differences in product length distribution. These data suggest charge transport is involved in primer length counting and may signal for primer template handoff to pol α.

Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron-sulfur clusters.

Biological assembly of iron-sulfur (Fe-S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA-hscBA-fdx-iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe-S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe-S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe-S cluster-containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron-transfer Fe-S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe-S scaffold protein responsible for the de novo Fe-S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe-S cluster biosynthesis.

The iron-binding CyaY and IscX proteins assist the ISC-catalyzed Fe-S biogenesis in Escherichia coli.

In eukaryotes, frataxin deficiency (FXN) causes severe phenotypes including loss of iron-sulfur (Fe-S) cluster protein activity, accumulation of mitochondrial iron and leads to the neurodegenerative disease Friedreich's ataxia. In contrast, in prokaryotes, deficiency in the FXN homolog, CyaY, was reported not to cause any significant phenotype, questioning both its importance and its actual contribution to Fe-S cluster biogenesis. Because FXN is conserved between eukaryotes and prokaryotes, this surprising discrepancy prompted us to reinvestigate the role of CyaY in Escherichia coli. We report that CyaY (i) potentiates E. coli fitness, (ii) belongs to the ISC pathway catalyzing the maturation of Fe-S cluster-containing proteins and (iii) requires iron-rich conditions for its contribution to be significant. A genetic interaction was discovered between cyaY and iscX, the last gene of the isc operon. Deletion of both genes showed an additive effect on Fe-S cluster protein maturation, which led, among others, to increased resistance to aminoglycosides and increased sensitivity to lambda phage infection. Together, these in vivo results establish the importance of CyaY as a member of the ISC-mediated Fe-S cluster biogenesis pathway in E. coli, like it does in eukaryotes, and validate IscX as a new bona fide Fe-S cluster biogenesis factor.

Novel Functions of an Iron-Sulfur Flavoprotein from Trichomonas vaginalis Hydrogenosomes

Iron-sulfur flavoproteins (Isf) are flavin mononucleotide (FMN)- and FeS cluster-containing proteins commonly encountered in anaerobic prokaryotes. However, with the exception of Isf from Methanosarcina thermophila, which participates in oxidative stress management by removing oxygen and hydrogen peroxide, none of these proteins has been characterized in terms of function. Trichomonas vaginalis, a sexually transmitted eukaryotic parasite of humans, was found to express several iron-sulfur flavoprotein (TvIsf) homologs in its hydrogenosomes. We show here that in addition to having oxygen-reducing activity, the recombinant TvIsf also functions as a detoxifying reductase of metronidazole and chloramphenicol, both of which are antibiotics effective against a variety of anaerobic microbes. TvIsf can utilize both NADH and reduced ferredoxin as electron donors. Given the prevalence of Isf in anaerobic prokaryotes, we propose that these proteins are central to a novel defense mechanism against xenobiotics.

A Mammalian Siderophore Synthesized by an Enzyme with a Bacterial Homolog Involved in Enterobactin Production

Intracellular iron homeostasis is critical for survival and proliferation. Lipocalin 24p3 is an iron-trafficking protein that binds iron through association with a bacterial siderophore, such as enterobactin, or a postulated mammalian siderophore. Here, we show that the iron-binding moiety of the 24p3-associated mammalian siderophore is 2,5-dihydroxybenzoic acid (2,5-DHBA), which is similar to 2,3-DHBA, the iron-binding component of enterobactin. We find that the murine enzyme responsible for 2,5-DHBA synthesis, BDH2, is the homolog of bacterial EntA, which catalyzes 2,3-DHBA production during enterobactin biosynthesis. RNA interference-mediated knockdown of BDH2 results in siderophore depletion. Mammalian cells lacking the siderophore accumulate abnormally high amounts of cytoplasmic iron, resulting in elevated levels of reactive oxygen species, whereas the mitochondria are iron deficient. Siderophore-depleted mammalian cells and zebrafish embryos fail to synthesize heme, an iron-dependent mitochondrial process. Our results reveal features of intracellular iron homeostasis that are conserved from bacteria through humans.

NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein.

The NifS and NifU proteins from Azotobacter vinelandii are required for the full activation of nitrogenase. NifS is a homodimeric cysteine desulfurase that supplies the inorganic sulfide necessary for formation of the Fe-S clusters contained within the nitrogenase component proteins. NifU has been suggested to complement NifS either by mobilizing the Fe necessary for nitrogenase Fe-S cluster formation or by providing an intermediate Fe-S cluster assembly site. As isolated, the homodimeric NifU protein contains one [2Fe-2S](2+, +) cluster per subunit, which is referred to as the permanent cluster. In this report, we show that NifU is able to interact with NifS and that a second, transient [2Fe-2S] cluster can be assembled within NifU in vitro when incubated in the presence of ferric ion, L-cysteine, and catalytic amounts of NifS. Approximately one transient [2Fe-2S] cluster is assembled per homodimer. The transient [2Fe-2S] cluster species is labile and rapidly released on reduction. We propose that transient [2Fe-2S] cluster units are formed on NifU and then released to supply the inorganic iron and sulfur necessary for maturation of the nitrogenase component proteins. The role of the permanent [2Fe-2S] clusters contained within NifU is not yet known, but they could have a redox function involving either the formation or release of transient [2Fe-2S] cluster units assembled on NifU. Because homologs to both NifU and NifS, respectively designated IscU and IscS, are found in non-nitrogen fixing organisms, it is possible that the function of NifU proposed here could represent a general mechanism for the maturation of Fe-S cluster-containing proteins.