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Mammalian ferredoxin 1 and 2 (FDX1/2) belong to an evolutionary conserved family of iron-sulfur cluster containing proteins and act as electron shutters between ferredoxin reductase (FDXR) and numerous proteins involved in critical biological pathways. FDX1 is involved in biogenesis of steroids and bile acids, Vitamin A/D metabolism, and lipoylation of tricarboxylic acid (TCA) cycle enzymes. FDX1 has been extensively characterized biochemically but its role in physiology and lipid metabolism has not been explored. In this study, we generated Fdx1-deficient mice and showed that knockout of both alleles of the Fdx1 gene led to embryonic lethality. We also showed that like Fdxr+/-+/-, Fdx1+/-+/- had a shorter life span and were prone to steatohepatitis. However, unlike Fdxr+/-+/-, Fdx1+/-+/- were not prone to spontaneous tumors. Additionally, we showed that FDX1 deficiency led to lipid droplet accumulation possibly via the ABCA1-SREBP1/2 pathway. Specifically, untargeted lipidomic analysis showed that FDX1 deficiency led to alterations in several classes of lipids, including cholesterol, triacylglycerides, acylcarnitines, ceramides, phospholipids and lysophospholipids. Taken together, our data indicate that FDX1 is essential for mammalian embryonic development and lipid homeostasis at both cellular and organismal levels.

Lung cancer, predominantly non-small cell lung cancer (NSCLC), remains a significant global health challenge, with limited therapeutic options for patients with KRAS-mutated tumors. Herein, a copper-based metal-organic framework (Cu-MOF) was applied as a novel cuproptosis-mediated nanoplatform for lung cancer therapy. Cu-MOF would disassemble and liberate copper ions under the acidic microenvironment of lysosomes of cancer cells, initiating a cascade of cellular events. The released copper ions catalyzes the Fenton reaction, generating hydroxyl radicals that induce oxidative damage, leading to cytoskeletal disruption and activation of caspase-3, ultimately triggering apoptosis. Simultaneously, with the mediation of the key regulatory factor FDX1, we found that the copper ions binding to the mitochondrial protein DLAT could result in the loss of iron-sulfur cluster proteins and aggregation of lipoylated proteins, which culminated in proteotoxic stress-induced cuproptosis. The pronounced anti-tumor effects of Cu-MOF with apoptosis and cuproptosis were confirmed both in vitro and in vivo experiments. Such dual induction of apoptosis and cuproptosis by Cu-MOF presents a promising therapeutic strategy for NSCLC, particularly for KRAS-mutated tumors, and expands potential applications of Cu-based nanomateirals for other cancers. [ABSTRACT FROM AUTHOR]

ImportanceThis work has broad relevance due to the ubiquity of dyes containing azo bonds in food and drugs. We report that azo dyes can be degraded by human gut bacteria through both enzymatic and nonenzymatic mechanisms, even from a single gut bacterial species. Furthermore, we revealed that environmental factors, oxygen, and L-Cysteine control the ability of E. coli to degrade azo dyes due to their impacts on bacterial transcription and metabolism. These results open up new opportunities to manipulate the azoreductase activity of the gut microbiome through the manipulation of host diet, suggest that azoreductase potential may be altered in patients suffering from gastrointestinal disease, and highlight the importance of studying bacterial enzymes for drug metabolism in their natural cellular and ecological context.

Copper is a trace element required by the organism, but once the level of copper exceeds the threshold, it becomes toxic and even causes death. The underlying mechanisms of copper-induced death are inconclusive, with different studies showing different opinions on the mechanism of copper-induced death. Multiple investigations have shown that copper induces oxidative stress, endoplasmic reticulum stress, nucleolar stress, and proteasome inhibition, all of which can result in cell death. The latest research elucidates a copper-dependent death and denominates it as cuproptosis. Cuproptosis takes place through the combination of copper and lipoylated proteins of the tricarboxylic acid cycle, triggering agglomeration of lipoylated proteins and loss of iron-sulfur cluster proteins, leading to proteotoxic stress and ultimately death. Given the toxicity and necessity of copper, abnormal levels of copper lead to diseases such as neurological diseases and cancer. The development of cancer has a high demand for copper, neurological diseases involve the change of copper contents and the binding of copper to proteins. There is a close relationship between these two kinds of diseases and copper. Here, we summarize the mechanisms of copper-related death, and the association between copper and diseases, to better figure out the influence of copper in cell death and diseases, thus advancing the clinical remedy of these diseases. [ABSTRACT FROM AUTHOR]

Copper is an essential element in the human body, involved in many physiological and metabolic functions, including coagulation, oxidative metabolism, and hormone production. The maintenance of copper homeostasis within cells is a complex procedure that is intrinsically controlled by a multitude of intricate mechanisms. Disorders of copper homeostasis encompass a wide range of pathological conditions, including degenerative neurological diseases, metabolic disorders, cardio-cerebrovascular diseases, and tumors. Cuproptosis, a recently identified non-apoptotic mode of cell death mode, is characterized by copper dependence and the regulation of mitochondrial respiration. Cuproptosis represents a novel form of cell death distinct from the previously described modes, including apoptosis, necrosis, pyroptosis, and ferroptosis. Excess copper has been shown to induce cuproptosis by stimulating protein toxic stress responses via copper-dependent abnormal oligomerization of lipoylation proteins within the tricarboxylic acid cycle and the subsequent reduction of iron-sulfur cluster protein levels. Ferredoxin1 facilitates the lipoacylation of dihydrolipoyl transacetylase, which in turn degrades iron-sulfur cluster proteins by reducing Cu2+ to Cu+, thereby inducing cell death. Furthermore, copper homeostasis is regulated by the copper transporter, and disturbances in this homeostasis result in cuproptosis. Current evidence suggests that cuproptosis plays an important role in the onset and development of several cardiovascular diseases. Copper-chelating agents, including ammonium tetrathiomolybdate (VI) and DL-penicillamine, have been shown to facilitate the alleviation of cardiovascular disease by inhibiting cuproptosis. It is hypothesized that oxidative phosphorylation inhibitors such as physical training may inhibit cuproptosis by inhibiting the protein stress response. In conclusion, the implementation of physical training may be a viable strategy to reducte the incidence of cuproptosis. [ABSTRACT FROM AUTHOR]

Prostate, bladder, and kidney cancers are the most common malignancies of the urinary system. Chemotherapeutic drugs are generally used as adjuvant treatment in the middle, late, or recurrence stages after surgery for urologic cancers. However, traditional chemotherapy is plagued by problems such as poor efficacy, severe side effects, and complications. Copper‐containing nanomedicines are promising novel cancer treatment modalities that can potentially overcome these disadvantages. Copper homeostasis and cuproptosis play crucial roles in the development, adaptability, and therapeutic sensitivity of urological malignancies. Cuproptosis refers to the direct binding of copper ions to lipoylated components of the tricarboxylic acid cycle, leading to protein oligomerization, loss of iron–sulfur proteins, proteotoxic stress, and cell death. This review focuses on copper homeostasis and cuproptosis as well as recent findings on copper and cuproptosis in urological malignancies. Furthermore, we highlight the potential therapeutic applications of copper‐ and cuproptosis‐targeted therapies to better understand cuproptosis‐based drugs for the treatment of urological tumors in the future. [ABSTRACT FROM AUTHOR]

Magnetic sense, or termed magnetoreception, has evolved in a broad range of taxa within the animal kingdom to facilitate orientation and navigation. MagRs, highly conserved A-type iron-sulfur proteins, are widely distributed across all phyla and play essential roles in both magnetoreception and iron-sulfur cluster biogenesis. However, the evolutionary origins and functional diversification of MagRs from their prokaryotic ancestor remain unclear. In this study, MagR sequences from 131 species, ranging from bacteria to humans, were selected for analysis, with 23 representative sequences covering species from prokaryotes to Mollusca, Arthropoda, Osteichthyes, Reptilia, Aves, and mammals chosen for protein expression and purification. Biochemical studies revealed a gradual increase in total iron content in MagRs during evolution. Three types of MagRs were identified, each with distinct iron and/or iron-sulfur cluster binding capacity and protein stability, indicating continuous expansion of the functional roles of MagRs during speciation and evolution. This evolutionary biochemical study provides valuable insights into how evolution shapes the physical and chemical properties of biological molecules such as MagRs and how these properties influence the evolutionary trajectories of MagRs. [ABSTRACT FROM AUTHOR]

Background: Somatic copy number alterations are a hallmark of cancer that offer unique opportunities for therapeutic exploitation. Here, we focused on the identification of specific vulnerabilities for tumors harboring chromosome 8p deletions. Methods: We developed and applied an integrative analysis of The Cancer Genome Atlas (TCGA), the Cancer Dependency Map (DepMap), and the Cancer Cell Line Encyclopedia to identify chromosome 8p-specific vulnerabilities. We employ orthogonal gene targeting strategies, both in vitro and in vivo, including short hairpin RNA-mediated gene knockdown and CRISPR/Cas9-mediated gene knockout to validate vulnerabilities. Results: We identified SLC25A28 (also known as MFRN2), as a specific vulnerability for tumors harboring chromosome 8p deletions. We demonstrate that vulnerability towards MFRN2 loss is dictated by the expression of its paralog, SLC25A37 (also known as MFRN1), which resides on chromosome 8p. In line with their function as mitochondrial iron transporters, MFRN1/2 paralog protein deficiency profoundly impaired mitochondrial respiration, induced global depletion of iron-sulfur cluster proteins, and resulted in DNA-damage and cell death. MFRN2 depletion in MFRN1-deficient tumors led to impaired growth and even tumor eradication in preclinical mouse xenograft experiments, highlighting its therapeutic potential. Conclusions: Our data reveal MFRN2 as a therapeutic target of chromosome 8p deleted cancers and nominate MFNR1 as the complimentary biomarker for MFRN2-directed therapies. [ABSTRACT FROM AUTHOR]

Summary: Root‐knot nematodes (RKN; Meloidogyne species) are plant pathogens that introduce several effectors in their hosts to facilitate infection. The actual targets and functioning mechanism of these effectors largely remain unexplored. This study illuminates the role and interplay of the Meloidogyne javanica nematode effector ROS suppressor (Mj‐NEROSs) within the host plant environment.Mj‐NEROSs suppresses INF1‐induced cell death as well as flg22‐induced callose deposition and reactive oxygen species (ROS) production. A transcriptome analysis highlighted the downregulation of ROS‐related genes upon Mj‐NEROSs expression. NEROSs interacts with the plant Rieske's iron–sulfur protein (ISP) as shown by yeast‐two‐hybrid and bimolecular fluorescence complementation.Secreted from the subventral pharyngeal glands into giant cells, Mj‐NEROSs localizes in the plastids where it interacts with ISP, subsequently altering electron transport rates and ROS production. Moreover, our results demonstrate that isp Arabidopsis thaliana mutants exhibit increased susceptibility to M. javanica, indicating ISP importance for plant immunity.The interaction of a nematode effector with a plastid protein highlights the possible role of root plastids in plant defense, prompting many questions on the details of this process. [ABSTRACT FROM AUTHOR]

In the field of human health research, the homeostasis of copper (Cu) is receiving increased attention due to its connection to pathological conditions, including diabetes mellitus (DM). Recent studies have demonstrated that proteins associated with Cu homeostasis, such as ATOX1, FDX1, ATP7A, ATPB, SLC31A1, p53, and UPS, also contribute to DM. Cuproptosis, characterized by Cu homeostasis dysregulation and Cu overload, has been found to cause the oligomerization of lipoylated proteins in mitochondria, loss of iron–sulfur protein, depletion of glutathione, production of reactive oxygen species, and cell death. Further research into how cuproptosis affects DM is essential to uncover its mechanism of action and identify effective interventions. In this article, we review the molecular mechanism of Cu homeostasis and the role of cuproptosis in the pathogenesis of DM. The study of small-molecule drugs that affect these proteins offers the possibility of moving from symptomatic treatment to treating the underlying causes of DM. [ABSTRACT FROM AUTHOR]

Background Age is the greatest risk factor for lower urinary tract symptoms attributed to benign prostatic hyperplasia (LUTS/BPH). Although LUTS/BPH can be managed with pharmacotherapy, treatment failure has been putatively attributed to numerous pathological features of BPH (eg, prostatic fibrosis, inflammation). Mitochondrial dysfunction is a hallmark of aging; however, its impact on the pathological features of BPH remains unknown. Methods Publicly available gene array data were analyzed. Immunohistochemistry examined mitochondrial proteins in the human prostate. The effect of complex I inhibition (rotenone) on a prostatic cell line was examined using quantitative polymerase chain reaction, immunocytochemistry, and Seahorse assays. Oleic acid (OA) was tested as a bypass of complex I inhibition. Aged mice were treated with OA to examine its effects on urinary dysfunction. Voiding was assessed longitudinally, and a critical complex I protein measured. Results Mitochondrial function and fibrosis genes were altered in BPH. Essential mitochondrial proteins (ie, voltage-dependent anion channels 1 and 2, PTEN-induced kinase 1, and NADH dehydrogenase [ubiquinone] iron–sulfur protein 3, mitochondrial [NDUFS3]) were significantly (p  < .05) decreased in BPH. Complex I inhibition in cultured cells resulted in decreased respiration, altered NDUFS3 expression, increased collagen deposition, and gene expression. OA ameliorated these effects. OA-treated aged mice had significantly (p  < .05) improved voiding function and higher prostatic NDUFS3 expression. Conclusions Complex I dysfunction is a potential contributor to fibrosis and lower urinary tract dysfunction in aged mice. OA partially bypasses complex I inhibition and therefore should be further investigated as a mitochondrial modulator for treatment of LUTS/BPH. Hypotheses generated in this investigation offer a heretofore unexplored cellular target of interest for the management of LUTS/BPH. [ABSTRACT FROM AUTHOR]

The human AAA-ATPase Bcs1L translocates the fully assembled Rieske iron-sulfur protein (ISP) precursor across the mitochondrial inner membrane, enabling respiratory Complex III assembly. Exactly how the folded substrate is bound to and released from Bcs1L has been unclear, and there has been ongoing debate as to whether subunits of Bcs1L act in sequence or in unison hydrolyzing ATP when moving the protein cargo. Here, we captured Bcs1L conformations by cryo-EM during active ATP hydrolysis in the presence or absence of ISP substrate. In contrast to the threading mechanism widely employed by AAA proteins in substrate translocation, subunits of Bcs1L alternate uniformly between ATP and ADP conformations without detectable intermediates that have different, co-existing nucleotide states, indicating that the subunits act in concert. We further show that the ISP can be trapped by Bcs1 when its subunits are all in the ADP-bound state, which we propose to be released in the apo form. How folded proteins are translocated across membranes is unknown for eukaryotes. Here, the authors propose a concerted translocation mechanism used by the AAA-ATPase Bcs1 to retro-translocate fully folded Rieske iron-sulfur protein across mitochondrial inner membrane. [ABSTRACT FROM AUTHOR]

Mitochondria play a central role in muscle metabolism and function. A unique family of iron-sulfur proteins, termed CDGSH Iron Sulfur Domain-containing (CISD/NEET) proteins, support mitochondrial function in skeletal muscles. The abundance of these proteins declines during aging leading to muscle degeneration. Although the function of the outer mitochondrial CISD/NEET proteins, CISD1/mitoNEET and CISD2/NAF-1, has been defined in skeletal muscle cells, the role of the inner mitochondrial CISD protein, CISD3/MiNT, is currently unknown. Here, we show that CISD3 deficiency in mice results in muscle atrophy that shares proteomic features with Duchenne muscular dystrophy. We further reveal that CISD3 deficiency impairs the function and structure of skeletal muscles, as well as their mitochondria, and that CISD3 interacts with, and donates its [2Fe-2S] clusters to, complex I respiratory chain subunit NADH Ubiquinone Oxidoreductase Core Subunit V2 (NDUFV2). Using coevolutionary and structural computational tools, we model a CISD3-NDUFV2 complex with proximal coevolving residue interactions conducive of [2Fe-2S] cluster transfer reactions, placing the clusters of the two proteins 10 to 16 Å apart. Taken together, our findings reveal that CISD3/MiNT is important for supporting the biogenesis and function of complex I, essential for muscle maintenance and function. Interventions that target CISD3 could therefore impact different muscle degeneration syndromes, aging, and related conditions. [ABSTRACT FROM AUTHOR]

17β-estradiol (E2) is a natural endocrine disruptor that is frequently detected in surface and groundwater sources, thereby threatening ecosystems and human health. The newly isolated E2-degrading strain Sphingomonas colocasiae C3-2 can degrade E2 through both the 4,5-seco pathway and the 9,10-seco pathway; the former is the primary pathway supporting the growth of this strain and the latter is a branching pathway. The novel gene cluster ean was found to be responsible for E2 degradation through the 4,5-seco pathway, where E2 is converted to estrone (E1) by EanA, which belongs to the short-chain dehydrogenases/reductases (SDR) superfamily. A three-component oxygenase system (including the P450 monooxygenase EanB1, the small iron-sulfur protein ferredoxin EanB2, and the ferredoxin reductase EanB3) was responsible for hydroxylating E1 to 4-hydroxyestrone (4-OH-E1). The enzymatic assay showed that the proportion of the three components is critical for its function. The dioxygenase EanC catalyzes ring A cleavage of 4-OH-E1, and the oxidoreductase EanD is responsible for the decarboxylation of the ring A-cleavage product of 4-OH-E1. EanR, a TetR family transcriptional regulator, acts as a transcriptional repressor of the ean cluster. The ean cluster was also found in other reported E2-degrading sphingomonads. In addition, the novel two-component monooxygenase EanE1E2 can open ring B of 4-OH-E1 via the 9,10-seco pathway, but its encoding genes are not located within the ean cluster. These results refine research on genes involved in E2 degradation and enrich the understanding of the cleavages of ring A and ring B of E2. [ABSTRACT FROM AUTHOR]

Iron-sulfur clusters are essential cofactors for proteins involved in various biological processes, such as electron transport, biosynthetic reactions, DNA repair, and gene expression regulation. Iron-sulfur cluster assembly protein IscA1 (or MagR) is found within the mitochondria of most eukaryotes. Magnetoreceptor (MagR) is a highly conserved A-type iron and iron-sulfur cluster-binding protein, characterized by two distinct types of iron-sulfur clusters, [2Fe-2S] and [3Fe-4S], each conferring unique magnetic properties. MagR forms a rod-like polymer structure in complex with photoreceptive cryptochrome (Cry) and serves as a putative magnetoreceptor for retrieving geomagnetic information in animal navigation. Although the N-terminal sequences of MagR vary among species, their specific function remains unknown. In the present study, we found that the N-terminal sequences of pigeon MagR, previously thought to serve as a mitochondrial targeting signal (MTS), were not cleaved following mitochondrial entry but instead modulated the efficiency with which iron-sulfur clusters and irons are bound. Moreover, the N-terminal region of MagR was required for the formation of a stable MagR/Cry complex. Thus, the N-terminal sequences in pigeon MagR fulfil more important functional roles than just mitochondrial targeting. These results further extend our understanding of the function of MagR and provide new insights into the origin of magnetoreception from an evolutionary perspective. [ABSTRACT FROM AUTHOR]

The high mortality rate in patients with chronic obstructive pulmonary disease (COPD) may be due to pulmonary hypertension (PH). These diseases are highly associated with cigarette smoke and its key component nicotine. Here, we created a novel animal model of PH using coexposure to nicotine (or cigarette smoke) and hypoxia. This heretofore unreported model showed significant early-onset pulmonary vasoremodeling and PH. Using newly generated mice with complementary smooth muscle–specific Rieske iron-sulfur protein (RISP) gene knockout and overexpression, we demonstrate that RISP is critically involved in promoting pulmonary vasoremodeling and PH, which are implemented by oxidative ataxia telangiectasia-mutated–mediated DNA damage and NF-κB–dependent inflammation in a reciprocal positive mechanism. Together, our findings establish for the first time an animal model of hypoxia-induced early-onset PH in which mitochondrial RISP-dependent DNA damage and NF-κB inflammation play critical roles in vasoremodeling. Specific therapeutic targets for RISP and related oxidative stress–associated signaling pathways may create unique and effective treatments for PH, chronic obstructive pulmonary disease, and their complications. [ABSTRACT FROM AUTHOR]

Target identification remains a critical challenge in inorganic drug discovery to deconvolute potential polypharmacology. Herein, we describe an improved approach to prioritize candidate protein targets based on a combination of dose-dependent chemoproteomics and treatment effects in living cancer cells for the rhenium tricarbonyl compound TRIP. Chemoproteomics revealed 89 distinct dose-dependent targets with concentrations of competitive saturation between 0.1 and 32 μM despite the broad proteotoxic effects of TRIP. Target-response networks revealed two highly probable targets of which the Fe-S cluster biogenesis factor NUBP2 was competitively saturated by free TRIP at nanomolar concentrations. Importantly, TRIP treatment led to a down-regulation of Fe-S cluster containing proteins and upregulated ferritin. Fe-S cluster depletion was further verified by assessing mitochondrial bioenergetics. Consequently, TRIP emerges as a first-in-class modulator of the scaffold protein NUBP2, which disturbs Fe-S cluster biogenesis at sub-cytotoxic concentrations in ovarian cancer cells.

The mechanisms underlying metabolic adaptation of pancreatic ductal adenocarcinoma (PDA) cells to pharmacologic inhibition of RAS-MAPK signaling are largely unknown. Using transcriptome and chromatin immunoprecipitation profiling of PDA cells treated with the MEK inhibitor (MEKi) trametinib, we identify transcriptional antagonism between c-MYC and the master transcription factors for lysosome gene expression, the MiT/TFE proteins. Under baseline conditions, c-MYC and MiT/TFE factors compete for binding to lysosome gene promoters to fine-tune gene expression. Treatment of PDA cells or patient organoids with MEKi leads to c-MYC downregulation and increased MiT/TFE-dependent lysosome biogenesis. Quantitative proteomics of immunopurified lysosomes uncovered reliance on ferritinophagy, the selective degradation of the iron storage complex ferritin, in MEKi-treated cells. Ferritinophagy promotes mitochondrial iron-sulfur cluster protein synthesis and enhanced mitochondrial respiration. Accordingly, suppressing iron utilization sensitizes PDA cells to MEKi, highlighting a critical and targetable reliance on lysosome-dependent iron supply during adaptation to KRAS-MAPK inhibition.SignificanceReduced c-MYC levels following MAPK pathway suppression facilitate the upregulation of autophagy and lysosome biogenesis. Increased autophagy-lysosome activity is required for increased ferritinophagy-mediated iron supply, which supports mitochondrial respiration under therapy stress. Disruption of ferritinophagy synergizes with KRAS-MAPK inhibition and blocks PDA growth, thus highlighting a key targetable metabolic dependency. See related commentary by Jain and Amaravadi, p. 2023. See related article by Santana-Codina et al., p. 2180. This article is highlighted in the In This Issue feature, p. 2007.

Elevated Susceptibility to Exogenous Seizure Triggers and Impaired Interneuron Excitability in a Mouse Model of Leigh Syndrome Epilepsy Manning A, Han V, Stephens A, Wang R, Bush N, Bard M, Ramirez JM, and Kalume F. Neurobiol Dis. 2023;187:106288 Mutations in the nicotinamide adenine dinucleotide dehydrogenase (ubiquinone reductase) iron-sulfur protein 4 (NDUFS4) gene, which encodes for a key structural subunit of the OXFOS complex I, lead to the most common form of mitochondrial disease in children known as Leigh syndrome (LS). As in other mitochondrial diseases, epileptic seizures constitute one of the most significant clinical features of LS. These seizures are often very difficult to treat and are a sign of poor disease prognosis. Mice with whole-body Ndufs4 KO are a well-validated model of LS; they exhibit epilepsy and several other clinical features of LS. We have previously shown that mice with Ndufs4 KO in only GABAergic interneurons (Gad2- Ndufs4 -KO) reproduce the severe epilepsy phenotype observed in the global KO mice. This observation indicated that these mice represent an excellent model of LS epilepsy isolated from other clinical manifestations of the disease. To further characterize this epilepsy phenotype, we investigated seizure susceptibility to selected exogenous seizure triggers in Gad2- Ndufs4 -KO mice. Then, using electrophysiology, imaging, and immunohistochemistry, we studied the cellular, physiological, and neuroanatomical consequences of Ndufs4 KO in GABAergic interneurons. Homozygous KO of Ndufs4 in GABAergic interneurons leads to a prominent susceptibility to exogenous seizure triggers, impaired interneuron excitability, and interneuron loss. Finally, we found that the hippocampus and cortex participate in the generation of seizure activity in Gad2- Ndufs4 -KO mice. These findings further define the LS epilepsy phenotype and provide important insights into the cellular mechanisms underlying epilepsy in LS and other mitochondrial diseases. [ABSTRACT FROM AUTHOR]

The cytosolic iron-sulfur (Fe-S) cluster assembly (CIA) pathway delivers Fe-S clusters to nuclear and cytosolic Fe-S proteins involved in essential cellular functions. Although the delivery process is regulated by the availability of iron and oxygen, it remains unclear how CIA components orchestrate the cluster transfer under varying cellular environments. Here, we utilized a targeted proteomics assay for monitoring CIA factors and substrates to characterize the CIA machinery. We find that nucleotide-binding protein 1 (NUBP1/NBP35), cytosolic iron-sulfur assembly component 3 (CIAO3/NARFL), and CIA substrates associate with nucleotide-binding protein 2 (NUBP2/CFD1), a component of the CIA scaffold complex. NUBP2 also weakly associates with the CIA targeting complex (MMS19, CIAO1, and CIAO2B) indicating the possible existence of a higher order complex. Interactions between CIAO3 and the CIA scaffold complex are strengthened upon iron supplementation or low oxygen tension, while iron chelation and reactive oxygen species weaken CIAO3 interactions with CIA components. We further demonstrate that CIAO3 mutants defective in Fe-S cluster binding fail to integrate into the higher order complexes. However, these mutants exhibit stronger associations with CIA substrates under conditions in which the association with the CIA targeting complex is reduced suggesting that CIAO3 and CIA substrates may associate in complexes independently of the CIA targeting complex. Together, our data suggest that CIA components potentially form a metabolon whose assembly is regulated by environmental cues and requires Fe-S cluster incorporation in CIAO3. These findings provide additional evidence that the CIA pathway adapts to changes in cellular environment through complex reorganization.

Viruses rely on host cells to replicate their genomes and assemble new viral particles. Thus, they have evolved intricate mechanisms to exploit host factors. Host cells, in turn, have developed strategies to inhibit viruses, resulting in a nuanced interplay of co-evolution between virus and host. This dynamic often involves competition for resources crucial for both host cell survival and virus replication. Iron and iron-containing cofactors, including iron–sulfur clusters, are known to be a heavily fought for resource during bacterial infections, where control over iron can tug the war in favor of the pathogen or the host. It is logical to assume that viruses also engage in this competition. Surprisingly, our knowledge about how viruses utilize iron (Fe) and iron–sulfur (FeS) clusters remains limited. The handful of reviews on this topic primarily emphasize the significance of iron in supporting the host immune response against viral infections. The aim of this review, however, is to organize our current understanding of how viral proteins utilize FeS clusters, to give perspectives on what questions to ask next and to propose important avenues for future investigations. [ABSTRACT FROM AUTHOR]

Iron-sulfur [Fe-S] clusters, comprising coordinated iron and sulfur atoms arranged in diverse configurations, play a pivotal role in redox reactions and various biological processes. Diverse structural variants of [Fe-S] clusters exist, each possessing distinct attributes and functions. Recent discovery of [Fe-S] clusters in infectious pathogens, such as Mycobacterium tuberculosis, and in viruses, such as rotavirus, polyomavirus, hepatitis virus, mimivirus, and coronavirus, have sparked interest in them being a potential therapeutics target. Recent findings have associated these [Fe-S] cluster proteins playing a critical role in structural and host protein activity. However, for a very long time, metalloenzymes containing iron-sulfur clusters have been prone to destabilization in the presence of oxygen, which led to a delayed understanding of [Fe-S] proteins compared to other non-heme iron-containing proteins. Consequently, working with [Fe-S] proteins require specialized equipment, such as anaerobic chambers to maintain cofactor integrity, and tools like ultraviolet visible (UV-Vis) spectroscopy, mass spectrometry, X-ray crystallography, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), Mössbauer spectroscopy and electrochemical characterization. Many of these [Fe-S] cluster proteins have been misannotated as Zinc-binding proteins when purified aerobically. Moreover, the assembly of these iron-sulfur cluster cofactors have not been fully understood since it is a multi-step assembly process. Additionally, disruptions in this assembly process have been linked to human diseases. With rapid advancements in anaerobic gloveboxes and spectroscopic techniques, characterization of these [Fe-S] cluster-containing proteins that are essential for the pathogens can open up new avenues for diagnostics and therapeutics. [ABSTRACT FROM AUTHOR]

Copper is an essential element for critical cellular functions such as mitochondrial respiration, cholesterol biosynthesis and immune response. Altered copper homeostasis has been associated with various disorders, including cancer. The copper overload is known to contribute to tumorigenesis, angiogenesis and metastasis, and recently it has been suggested that the elevated level of this element may also create vulnerability to a novel cell death mechanism, named cuproptosis. Excessive amount of copper in mitochondria binds to lipoylated enzymes of the TCA cycle and forms insoluble oligomers. The aggregation of these oligomers and subsequent iron–sulfur cluster protein loss results in proteotoxic stress and eventual cell death. Hepatocellular carcinoma is a common malignancy with a low survival rate, despite the available treatment options. The discovery of cuproptosis led many researchers to explore its potential use in hepatocellular cancer therapy due to the rich mitochondria content of hepatic cells. In this regard, a number of genomic studies were conducted to discover several cuproptosis-related genes and explored their association with prognosis, survival and immunotherapy response. This review brings together the available data on the relationship between cuproptosis and hepatocellular cancer for the first time, and highlights some of the potential biomarkers or target molecules that may be useful in the treatment. [ABSTRACT FROM AUTHOR]

The robustness of NMR coherence transfer in proximity of a paramagnetic center depends on the relaxation properties of the nuclei involved. In the case of Iron-Sulfur Proteins, different pulse schemes or different parameter sets often provide complementary results. Tailored versions of HCACO and CACO experiments significantly increase the number of observed Cα/C' connectivities in highly paramagnetic systems, by recovering many resonances that were lost due to paramagnetic relaxation. Optimized 13C direct detected experiments can significantly extend the available assignments, improving the overall knowledge of these systems. The different relaxation properties of Cα and C' nuclei are exploited in CACO vs COCA experiments and the complementarity of the two experiments is used to obtain structural information. The two [Fe2S2]+ clusters containing NEET protein CISD3 and the one [Fe4S4]2+ cluster containing HiPIP protein PioC have been taken as model systems. We show that tailored experiments contribute to decrease the blind sphere around the cluster, to extend resonance assignment of cluster bound cysteine residues and to retrieve details on the topology of the iron-bound ligand residues. [ABSTRACT FROM AUTHOR]

The Fe protein of nitrogenase plays multiple roles in substrate reduction and cluster maturation via its redox-active [Fe4 S4 ] cluster. Here we report the synthesis and characterization of a water-soluble [Fe4 Se4 ] cluster that is used to substitute the [Fe4 S4 ] cluster of the Azotobacter vinelandii Fe protein (AvNifH). Biochemical, EPR and XAS/EXAFS analyses demonstrate the ability of the [Fe4 Se4 ] cluster to adopt the super-reduced, all-ferrous state upon its incorporation into AvNifH. Moreover, these studies reveal that the [Fe4 Se4 ] cluster in AvNifH already assumes a partial all-ferrous state ([Fe4 Se4 ]0 ) in the presence of dithionite, where its [Fe4 S4 ] counterpart in AvNifH exists solely in the reduced state ([Fe4 S4 ]1+ ). Such a discrepancy in the redox properties of the AvNifH-associated [Fe4 Se4 ] and [Fe4 S4 ] clusters can be used to distinguish the differential redox requirements for the substrate reduction and cluster maturation of nitrogenase, pointing to the utility of chalcogen-substituted FeS clusters in future mechanistic studies of nitrogenase catalysis and assembly.

Iron-sulfur cluster proteins are involved in critical functions for gene expression regulation and mitochondrial bioenergetics including the oxidative phosphorylation system. The c.215G>A p.(Arg72Gln) variant in NFS1 has been previously reported to cause infantile mitochondrial complex II and III deficiency. We describe three additional unrelated patients with the same missense variant. Two infants with the same homozygous variant presented with hypotonia, weakness and lactic acidosis, and one patient with compound heterozygous p.(Arg72Gln) and p.(Arg412His) variants presented as a young adult with gastrointestinal symptoms and fatigue. Skeletal muscle biopsy from patients 1 and 3 showed abnormal mitochondrial morphology, and functional analyses demonstrated decreased activity in respiratory chain complex II and variably in complexes I and III. We found decreased mitochondrial and cytosolic aconitase activities but only mildly affected lipoylation of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase enzymes. Our studies expand the phenotypic spectrum and provide further evidence for the pathogenicity and functional sequelae of NFS1-related disorders with disturbances in both mitochondrial and cytosolic iron-sulfur cluster containing enzymes.

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H2. The biosynthesis of the [2Fe]H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO)2(CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe2(μ-SH)2(CN)2(CO)4]2- active HydA1 can be biosynthesized without maturases HydG and HydE.

Molybdenum nitrogenase catalyses the reduction of N2 to NH3 at its cofactor, an [(R-homocitrate)MoFe7S9C] cluster synthesized via the formation of a [Fe8S9C] L-cluster prior to the insertion of molybdenum and homocitrate. We have previously identified a [Fe8S8C] L*-cluster, which is homologous to the core structure of the L-cluster but lacks the 'ninth sulfur' in the belt region. However, direct evidence and mechanistic details of the L*- to L-cluster conversion upon 'ninth sulfur' insertion remain elusive. Here we trace the 'ninth sulfur' insertion using SeO32- and TeO32- as 'labelled' SO32-. Biochemical, electron paramagnetic resonance and X-ray absorption spectroscopy/extended X-ray absorption fine structure studies suggest a role of the 'ninth sulfur' in cluster transfer during cofactor biosynthesis while revealing the incorporation of Se2-- and Te2--like species into the L-cluster. Density functional theory calculations further point to a plausible mechanism involving in situ reduction of SO32- to S2-, thereby suggesting the utility of this reaction to label the catalytically important belt region for mechanistic investigations of nitrogenase.

The [FeFe] hydrogenase catalyzes the redox interconversion of protons and H2 with a Fe-S "H-cluster" employing CO, CN, and azadithiolate ligands to two Fe centers. The biosynthesis of the H-cluster is a highly interesting reaction carried out by a set of Fe-S maturase enzymes called HydE, HydF, and HydG. HydG, a member of the radical S-adenosylmethionine (rSAM) family, converts tyrosine, cysteine, and Fe(II) into an organometallic Fe(II)(CO)2(CN)cysteine "synthon", which serves as the substrate for HydE. Although key aspects of the HydG mechanism have been experimentally determined via isotope-sensitive spectroscopic methods, other important mechanistic questions have eluded experimental determination. Here, we use computational quantum chemistry to refine the mechanism of the HydG catalytic reaction. We utilize quantum mechanics/molecular mechanics simulations to investigate the reactions at the canonical Fe-S cluster, where a radical cleavage of the tyrosine substrate takes place and proceeds through a relay of radical intermediates to form HCN and a COO•- radical anion. We then carry out a broken-symmetry density functional theory study of the reactions at the unusual five-iron auxiliary Fe-S cluster, where two equivalents of CN- and COOH• coordinate to the fifth "dangler iron" in a series of substitution and redox reactions that yield the synthon as the final product for further processing by HydE.

The eukaryotic cytosolic Fe-S protein assembly (CIA) machinery inserts iron-sulfur (Fe-S) clusters into cytosolic and nuclear proteins. In the final maturation step, the Fe-S cluster is transferred to the apo-proteins by the CIA-targeting complex (CTC). However, the molecular recognition determinants of client proteins are unknown. We show that a conserved [LIM]-[DES]-[WF]-COO- tripeptide is present at the C-terminus of more than a quarter of clients or their adaptors. When present, this targeting complex recognition (TCR) motif is necessary and sufficient for binding to the CTC in vitro and for directing Fe-S cluster delivery in vivo. Remarkably, fusion of this TCR signal enables engineering of cluster maturation on a nonnative protein via recruitment of the CIA machinery. Our study advances our understanding of Fe-S protein maturation and paves the way for bioengineering novel pathways containing Fe-S enzymes. [ABSTRACT FROM AUTHOR]

Background: Adaptation to alkalinization of the medium in fungi involves an extensive remodeling of gene expression. Komagataella phaffii is an ascomycetous yeast that has become an organism widely used for heterologous protein expression. We explore here the transcriptional impact of moderate alkalinization in this yeast, in search of suitable novel promoters able to drive transcription in response to the pH signal. Results: In spite of a minor effect on growth, shifting the cultures from pH 5.5 to 8.0 or 8.2 provokes significant changes in the mRNA levels of over 700 genes. Functional categories such as arginine and methionine biosynthesis, non-reductive iron uptake and phosphate metabolism are enriched in induced genes, whereas many genes encoding iron-sulfur proteins or members of the respirasome were repressed. We also show that alkalinization is accompanied by oxidative stress and we propose this circumstance as a common trigger of a subset of the observed changes. PHO89, encoding a Na+/Pi cotransporter, appears among the most potently induced genes by high pH. We demonstrate that this response is mainly based on two calcineurin-dependent response elements located in its promoter, thus indicating that alkalinization triggers a calcium-mediated signal in K. phaffii. Conclusions: This work defines in K. phaffii a subset of genes and diverse cellular pathways that are altered in response to moderate alkalinization of the medium, thus setting the basis for developing novel pH-controlled systems for heterologous protein expression in this fungus. [ABSTRACT FROM AUTHOR]

Bcs1, a homo-heptameric transmembrane AAA-ATPase, facilitates folded Rieske iron-sulfur protein translocation across the inner mitochondrial membrane. Structures in different nucleotide states (ATPγS, ADP, apo) provided conformational snapshots, but the kinetics and structural transitions of the ATPase cycle remain elusive. Here, using high-speed atomic force microscopy (HS-AFM) and line scanning (HS-AFM-LS), we characterized single-molecule Bcs1 ATPase cycling. While the ATP conformation had ~5600 ms lifetime, independent of the ATP-concentration, the ADP/apo conformation lifetime was ATP-concentration dependent and reached ~320 ms at saturating ATP-concentration, giving a maximum turnover rate of 0.17 s−1. Importantly, Bcs1 ATPase cycle conformational changes occurred in concert. Furthermore, we propose that the transport mechanism involves opening the IMS gate through energetically costly straightening of the transmembrane helices, potentially driving rapid gate resealing. Overall, our results establish a concerted ATPase cycle mechanism in Bcs1, distinct from other AAA-ATPases that use a hand-over-hand mechanism. Bcs1, a transmembrane AAA-ATPase, facilitates the translocation of folded ISP across the inner mitochondrial membrane. This study shows that the Bcs1 ATPase cycle conformational changes are highly concerted, unlike the canonical hand-over-hand mechanism. [ABSTRACT FROM AUTHOR]

Copper overload is a novel way to achieve copper‐ion‐interference therapy by disrupting copper homeostasis and treating diseases through multiple cell death pathways. However, it is difficult to reach copper overload since excess intracellular copper ions will be pumped out. Herein, copper overload is achieved by both raising cellular uptake and reducing the efflux of copper ions using hydrogen sulfide (H2S)‐responsive copper hydroxyphosphate nanoparticles (Cu2(PO4)(OH) NPs). After immersion in an H2S‐enriched colon cancer microenvironment, Cu2(PO4)(OH) NPs can transform into copper sulfide NPs with reduced size for higher cell entering, resulting in improved Fenton activity as well as copper ion dissociation. Reactive oxygen species generated by the Fenton reaction not only activate inflammasomes and Caspase‐1 proteins, cause the cleavage of gasdermin D to induce pyroptosis, but also affect mitochondrial function and down‐regulate copper exporter ATP7A to further reduce the copper excretion. The combination of higher endocytosis and lower exportation leads to maximized copper overload. Together with the efficient copper ions release, mitochondrial tricarboxylic acid cycle can be disrupted and iron‐sulfur cluster proteins are downregulated, ultimately triggering cuproptosis. As both pyroptosis and cuproptosis are efficient ways to induce cell death, this study provides a novel way to realize effective tumor‐targeted therapy based on H2S‐activated copper overload with simple Cu2(PO4)(OH) NPs. [ABSTRACT FROM AUTHOR]

Iron-sulfur clusters are prosthetic groups of proteins involved in various biological processes. However, details of the immature state of the iron-sulfur cluster into proteins have not yet been elucidated. We report here the first structural analysis of the Zn-containing form of a Rieske-type iron-sulfur protein, PetA, from Thermochromatium tepidum (TtPetA) by X-ray crystallography and small-angle X-ray scattering analysis. The Zn-containing form of TtPetA was indicated to be a dimer in solution. The zinc ion adopts a regular tetra-coordination with two chloride ions and two cysteine residues. Only a histidine residue in the cluster-binding site exhibited a conformational difference from the [2Fe-2S] containing form. The Zn-containing structure indicates that the conformation of the cluster binding site is already constructed and stabilized before insertion of [2Fe-2S]. The binding mode of ZnCl2, similar to the [2Fe-2S] cluster, suggests that the zinc ions might be involved in the insertion of the [2Fe-2S] cluster. Iron–sulfur clusters are important cofactors involved in a wide range of protein functions, however, the incorporation of FeS clusters into proteins remains underexplored. Here, the authors report the crystal structure of a Zn-containing form of a Rieske protein, in which ZnCl2 binds in an FeS-binding site, representing a putative immature form of a Rieske protein prior to FeS insertion. [ABSTRACT FROM AUTHOR]

[FeFe]-hydrogenases use a unique organometallic complex, termed the H cluster, to reversibly convert H2 into protons and low-potential electrons. It can be best described as a [Fe4S4] cluster coupled to a unique [2Fe]H center where the reaction actually takes place. The latter corresponds to two iron atoms, each of which is bound by one CN- ligand and one CO ligand. The two iron atoms are connected by a unique azadithiolate molecule (-S-CH2-NH-CH2-S-) and an additional bridging CO. This [2Fe]H center is built stepwise thanks to the well-orchestrated action of maturating enzymes that belong to the Hyd machinery. Among them, HydG converts l-tyrosine into CO and CN- to produce a unique l-cysteine-Fe(CO)2CN species termed complex-B. Very recently, HydE was shown to perform radical-based chemistry using synthetic complex-B as a substrate. Here we report the high-resolution crystal structure that establishes the identity of the complex-B-bound HydE. By triggering the reaction prior to crystallization, we trapped a new five-coordinate Fe species, supporting the proposal that HydE performs complex modifications of complex-B to produce a monomeric "SFe(CO)2CN" precursor to the [2Fe]H center. Substrate access, product release, and intermediate transfer are also discussed.

The radical S-adenosylmethionine (rSAM) enzyme SuiB catalyzes the formation of an unusual carbon-carbon bond between the sidechains of lysine (Lys) and tryptophan (Trp) in the biosynthesis of a ribosomal peptide natural product. Prior work on SuiB has suggested that the Lys-Trp cross-link is formed via radical electrophilic aromatic substitution (rEAS), in which an auxiliary [4Fe-4S] cluster (AuxI), bound in the SPASM domain of SuiB, carries out an essential oxidation reaction during turnover. Despite the prevalence of auxiliary clusters in over 165,000 rSAM enzymes, direct evidence for their catalytic role has not been reported. Here, we have used electron paramagnetic resonance (EPR) spectroscopy to dissect the SuiB mechanism. Our studies reveal substrate-dependent redox potential tuning of the AuxI cluster, constraining it to the oxidized [4Fe-4S]2+ state, which is active in catalysis. We further report the trapping and characterization of an unprecedented cross-linked Lys-Trp radical (Lys-Trp•) in addition to the organometallic Ω intermediate, providing compelling support for the proposed rEAS mechanism. Finally, we observe oxidation of the Lys-Trp• intermediate by the redox-tuned [4Fe-4S]2+ AuxI cluster by EPR spectroscopy. Our findings provide direct evidence for a role of a SPASM domain auxiliary cluster and consolidate rEAS as a mechanistic paradigm for rSAM enzyme-catalyzed carbon-carbon bond-forming reactions.

In the nematode Caenorhabditis elegans, signals derived from bacteria in the diet, the animal's major nutrient source, can modulate both behavior and healthspan. Here we describe a dual role for trimethylamine (TMA), a human gut flora metabolite, which acts as a nutrient signal and a neurotoxin. TMA and its associated metabolites are produced by the human gut microbiome and have been suggested to serve as risk biomarkers for diabetes and cardiovascular diseases. We demonstrate that the tyramine receptor TYRA-3, a conserved G protein-coupled receptor (GPCR), is required to sense TMA and mediate its responses. TMA activates guanylyl cyclase DAF-11 signaling through TYRA-3 in amphid neurons (ASK) and ciliated neurons (BAG) to mediate food-sensing behavior. Bacterial mutants deficient in TMA production enhance dauer formation, extend lifespan, and are less preferred as a food source. Increased levels of TMA lead to neural damage in models of Parkinson's disease and shorten lifespan. Our results reveal conserved signaling pathways modulated by TMA in C. elegans that are likely to be relevant for its effects in mammalian systems.

Although most organisms synthesize methionine from homocysteine and methyl folates, some have "core" methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

Iron-sulfur clusters play a central role in cellular function and are regulated by the ATM protein. Iron-sulfur clusters are part of the cellular sulfide pool, which functions to maintain cardiovascular health, and consists of free hydrogen sulfide, iron-sulfur clusters, protein bound sulfides, which constitute the total cellular sulfide fraction. ATM protein signaling and the drug pioglitazone share some cellular effects, which led us to examine the effects of this drug on cellular iron-sulfur cluster formation. Additionally, as ATM functions in the cardiovasculature and its signaling may be diminished in cardiovascular disease, we examined pioglitazone in the same cell type, with and without ATM protein expression. We examined the effects of pioglitazone treatment on the total cellular sulfide profile, the glutathione redox state, cystathionine gamma-lyase enzymatic activity, and on double-stranded DNA break formation in cells with and without ATM protein expression. Pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions and reduced cystathionine gamma-lyase enzymatic activity in cells with and without ATM protein expression. Interestingly, pioglitazone also increased reduced glutathione and lowered DNA damage in cells without ATM protein expression, but not in ATM wild-type cells. These results are interesting as the acid-labile (iron-sulfur cluster), bound sulfur cellular fractions, and reduced glutathione are low in cardiovascular disease. Here we found that pioglitazone increased the acid-labile (iron-sulfur cluster) and bound sulfur cellular fractions, impinges on hydrogen sulfide synthesis, and exerts beneficial effect on cells with deficient ATM protein signaling. Thus, we show a novel pharmacologic action for pioglitazone. • Pioglitazone increases cellular iron-sulfur complex and protein sulfhydration. • Pioglitazone impinges are several aspects of hydrogen sulfide function and synthesis. • Pioglitazone increases reduced glutathione levels and DNA repair capacity in ATM protein expression-deficient cells. • Pioglitazone is a possible pharmacologic treatment for ataxia-telangiectasia. [ABSTRACT FROM AUTHOR]

Copper, a transition metal, serves as an essential co-factor in numerous enzymatic active sites and constitutes a vital trace element in the human body, participating in crucial life-sustaining activities such as energy metabolism, antioxidation, coagulation, neurotransmitter synthesis, iron metabolism, and tetramer deposition. Maintaining the equilibrium of copper ions within biological systems is of paramount importance in the prevention of atherosclerosis and associated cardiovascular diseases. Copper induces cellular demise through diverse mechanisms, encompassing reactive oxygen species responses, apoptosis, necrosis, pyroptosis, and mitochondrial dysfunction. Recent research has identified and dubbed a novel regulatory cell death modality--"cuprotosis"--wherein copper ions bind to acylated proteins in the tricarboxylic acid cycle of mitochondrial respiration, resulting in protein aggregation, subsequent downregulation of iron-sulfur cluster protein expression, induction of proteotoxic stress, and eventual cell death. Scholars have synthesized copper complexes by combining copper ions with various ligands, exploring their significance and applications in cancer therapy. This review comprehensively examines the multiple pathways of copper metabolism, copper-induced regulatory cell death, and the current status of copper complexes in cancer treatment. [ABSTRACT FROM AUTHOR]

Iron–sulfur (Fe–S) clusters are ancient protein cofactors ubiquitously exist in organisms. They are involved in many important life processes. Plastids are semi-autonomous organelles with a double membrane and it is believed to originate from a cyanobacterial endosymbiont. By learning form the research in cyanobacteria, a Fe–S cluster biosynthesis and delivery pathway has been proposed and partly demonstrated in plastids, including iron uptake, sulfur mobilization, Fe–S cluster assembly and delivery. Fe–S clusters are essential for the downstream Fe–S proteins to perform their normal biological functions. Because of the importance of Fe–S proteins in plastid, researchers have made a lot of research progress on this pathway in recent years. This review summarizes the detail research progress made in recent years. In addition, the scientific problems remained in this pathway are also discussed. [ABSTRACT FROM AUTHOR]

Copper is an essential mineral nutrient that provides the cofactors for some key enzymes. However, excess copper is paradoxically cytotoxic. Wilson's disease is an autosomal recessive hereditary disease characterized by pathological copper accumulation in many organs, with high mortality and disability. Nevertheless, many questions about the molecular mechanism in Wilson's disease remain unknown and there is an imperative need to address these questions to better exploit therapeutic strategy. In this study, we constructed the mouse model of Wilson's disease, ATP7A−/− immortalized lymphocyte cell line and ATP7B knockdown cells to explore whether copper could impair iron-sulfur cluster biogenesis in eukaryotic mitochondria. Through a series of cellular, molecular, and pharmacological analyses, we demonstrated that copper could suppress the assembly of Fe–S cluster, decrease the activity of the Fe–S enzyme and disorder the mitochondrial function both in vivo and in vitro. Mechanistically, we found that human ISCA1, ISCA2 and ISCU proteins have a strong copper-binding activity, which would hinder the process of iron-sulfur assembly. Of note, we proposed a novel mechanism of action to explain the toxicity of copper by providing evidence that iron-sulfur cluster biogenesis may be a primary target of copper toxicity both in cells and mouse models. In summary, the current work provides an in-depth study on the mechanism of copper intoxication and describes a framework for the further understanding of impaired Fe–S assembly in the pathological processes of Wilson's diseases, which helps to develop latent therapeutic strategies for the management of copper toxicity. [Display omitted] • Copper could suppress the assembly of Fe–S cluster and decrease the activity of the Fe–S enzyme. • ISCA1, ISCA2 and ISCU have a copper-binding activity, which would hinder the process of iron-sulfur assembly. • Iron-sulfur cluster biogenesis is an important target of copper toxicity both in cells and mouse models. [ABSTRACT FROM AUTHOR]

Introduction: Hereditary necrotizing myelopathy (HNM) in young Kooiker dogs is characterized by progressive ataxia and paralysis with autosomal recessive inheritance. The basic genetic defect is unknown. We investigated the possible cause by a genome-wide analysis using six affected and 17 unrelated unaffected Kooiker dogs and by functional follow-up studies. Method: The HNM locus was mapped by a case–control study using a dense SNP array and confirmed by linkage analysis of two pedigrees. The gene exons in the critical region were analyzed by next-generation sequencing. The functional effect of the candidate canine IBA57 pathogenic variant was biochemically examined in an established HeLa cell culture model in which the endogenous IBA75 gene product was depleted by RNAi. Results: The basic defect was localized in the centromeric 5 Mb region of canine chromosome 14. The most associated SNP co-segregated fully with HNM and reached an LOD score of 6.1. A candidate pathogenic mutation was found in the iron–sulfur cluster assembly gene IBA57 and led to the amino acid substitution R147W. The expression of human IBA57 harboring the canine R147W exchange could only partially restore the biochemical defects of several mitochondrial [4Fe-4S] proteins upon IBA57 depletion, showing that the mutant protein is functionally impaired. Discussion: Pathogenic variants in human IBA57 cause multiple mitochondrial dysfunction syndrome 3 (MMDS3), a neurodegenerative disorder with distant similarities to HNM. The incomplete functional complementation of IBA57- depleted human cells by IBA57-R147W identifies the DNA mutation in affected Kooiker dogs as the genetic cause of HNM. Our findings further expand the phenotypic spectrum of pathogenic IBA57 variants. [ABSTRACT FROM AUTHOR]

Simple Summary: Rett syndrome (RTT) is a genetic disorder caused by mutations in the X-chromosome. These mutations distort the function of a protein (methyl-CpG-binding protein 2), which controls the expression of several other genes. The resulting pathology, which manifests mostly in female patients, is associated with a number of deficits in brain functioning and development. On the cellular level, the defective functioning of mitochondria—the powerplants of the cells—is one of several hallmarks of RTT. Mitochondria contain more than 1000 different proteins, and their composition differs among tissues. Our study aims to reveal differences between the mitochondrial proteomes of a mouse model of RTT and wild-type (WT) mice. Two brain regions were studied, the hippocampus and neocortex, both of which are pivotal for cognitive function. Indeed, numerous differences were identified, which are not only mouse strain (genotype)-specific but also brain-region-specific. These differentially expressed proteins could affect mitochondrial dynamics, oxidative phosphorylation, and other important aspects of mitochondrial functioning. The data obtained will contribute to improving our understanding of mitochondrial malfunctioning during the progression of RTT, and they will pave the way for further translational medicine research. Rett syndrome (RTT) is a genetic neurodevelopmental disorder with mutations in the X-chromosomal MECP2 (methyl-CpG-binding protein 2) gene. Most patients are young girls. For 7–18 months after birth, they hardly present any symptoms; later they develop mental problems, a lack of communication, irregular sleep and breathing, motor dysfunction, hand stereotypies, and seizures. The complex pathology involves mitochondrial structure and function. Mecp2−/y hippocampal astrocytes show increased mitochondrial contents. Neurons and glia suffer from oxidative stress, a lack of ATP, and increased hypoxia vulnerability. This spectrum of changes demands comprehensive molecular studies of mitochondria to further define their pathogenic role in RTT. Therefore, we applied a comparative proteomic approach for the first time to study the entity of mitochondrial proteins in a mouse model of RTT. In the neocortex and hippocampus of symptomatic male mice, two-dimensional gel electrophoresis and subsequent mass-spectrometry identified various differentially expressed mitochondrial proteins, including components of respiratory chain complexes I and III and the ATP-synthase FoF1 complex. The NADH-ubiquinone oxidoreductase 75 kDa subunit, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, NADH dehydrogenase [ubiquinone] flavoprotein 2, cytochrome b-c1 complex subunit 1, and ATP synthase subunit d are upregulated either in the hippocampus alone or both the hippocampus and neocortex of Mecp2−/y mice. Furthermore, the regulatory mitochondrial proteins mitofusin-1, HSP60, and 14-3-3 protein theta are decreased in the Mecp2−/y neocortex. The expressional changes identified provide further details of the altered mitochondrial function and morphology in RTT. They emphasize brain-region-specific alterations of the mitochondrial proteome and support the notion of a metabolic component of this devastating disorder. [ABSTRACT FROM AUTHOR]

Systematic studies have revealed interactions between components of the Hsp90 chaperone system and Fe/S protein biogenesis or iron regulation. In addition, two chloroplast‐localized DnaJ‐like proteins, DJA5 and DJA6, function as specific iron donors in plastidial Fe/S protein biogenesis. Here, we used Saccharomyces cerevisiae to study the impact of both the Hsp90 chaperone and the yeast DJA5‐DJA6 homologs, the essential cytosolic Ydj1, and the mitochondrial Mdj1, on cellular iron‐related processes. Despite severe phenotypes induced upon depletion of these crucial proteins, there was no critical in vivo impact on Fe/S protein biogenesis or iron regulation. Importantly, unlike the plant DJA5‐DJA6 iron chaperones, Ydj1 and Mdj1 did not bind iron in vivo, suggesting that these proteins use zinc for function under normal physiological conditions. [ABSTRACT FROM AUTHOR]

Aim To explore the pyroptosis of vascular smooth muscle cell (VSMC) induced by Chlamydia pneumoniae (C. pn) infection and its possible mechanisms. Methods Primary rat VSMC were cultured by explant method. After the model of VSMC infected with C. pn was established, the changes in morphology of VSMC were observed under an inverted phase microscope, the lactic dehydrogenase (LDH) content was detected by the kit, and the expression levels of GSDMD and Caspase-1 were determined by Western blot, the changes in mitochondrial oxidative phosphorylation and the expression of complex-related proteins were measured by quantitative proteomic analysis by tandem mass tag technology and gene ontology. Results Compared with the control group, bubble-like vesicles were found outside the membrane of VSMC after C. pn infection under an inverted phase microscope. After C. pn infection of VSMC for 36 h and 48 h, LDH content increased by 38. 92% and 79. 54% (P <0. 001), respectively, and the expression of pyropotosisrelated protein GSDMD increased by 1. 74 times and 1. 67 times (P<0. 001). After C. pn infection of VSMC for 48 h, the expression(pro-Caspase-1) and activity(Caspase-1 p12/p10)of Caspase-1 increased by 2. 69 times and 3. 47 times (P<0. 001), respectively. The mass spectrometry results showed that there were 20 differentially expressed proteins enriched in the oxidative phosphorylation pathway after C. pn infection, and at the same time, ComplexⅠubiquinone oxidoreductase iron-sulfur protein 4 (NDUFS4) decreased significantly. Further Western blot results showed that the expression level of NDUFS4 decreased by 57. 5% and 57% (P<0. 001) after C. pn infection of VSMC for 36 h and 48 h respectively. Conclusion C. pn infection may induce VSMC pyroptosis by affecting mitochondrial function through downregulating NDUFS4 expression. [ABSTRACT FROM AUTHOR]

Among the various components of the protozoan Plasmodium mitochondrial respiratory chain, only Complex III is a validated cellular target for antimalarial drugs. The compound CK-2-68 was developed to specifically target the alternate NADH dehydrogenase of the malaria parasite respiratory chain, but the true target for its antimalarial activity has been controversial. Here, we report the cryo-EM structure of mammalian mitochondrial Complex III bound with CK-2-68 and examine the structure-function relationships of the inhibitor's selective action on Plasmodium. We show that CK-2-68 binds specifically to the quinol oxidation site of Complex III, arresting the motion of the iron-sulfur protein subunit, which suggests an inhibition mechanism similar to that of Pf-type Complex III inhibitors such as atovaquone, stigmatellin, and UHDBT. Our results shed light on the mechanisms of observed resistance conferred by mutations, elucidate the molecular basis of the wide therapeutic window of CK-2-68 for selective action of Plasmodium vs. host cytochrome bc1, and provide guidance for future development of antimalarials targeting Complex III. [ABSTRACT FROM AUTHOR]

Rhodopseudomonas palustris is an alphaproteobacterium with impressive metabolic versatility, capable of oxidizing ferrous iron to fix carbon dioxide using light energy. Photoferrotrophic iron oxidation is one of the most ancient metabolisms, sustained by the pio operon coding for three proteins: PioB and PioA, which form an outer-membrane porin–cytochrome complex that oxidizes iron outside of the cell and transfers the electrons to the periplasmic high potential iron–sulfur protein (HIPIP) PioC, which delivers them to the light-harvesting reaction center (LH-RC). Previous studies have shown that PioA deletion is the most detrimental for iron oxidation, while, the deletion of PioC resulted in only a partial loss. The expression of another periplasmic HiPIP, designated Rpal_4085, is strongly upregulated in photoferrotrophic conditions, making it a strong candidate for a PioC substitute. However, it is unable to reduce the LH-RC. In this work we used NMR spectroscopy to map the interactions between PioC, PioA, and the LH-RC, identifying the key amino acid residues involved. We also observed that PioA directly reduces the LH-RC, and this is the most likely substitute upon PioC deletion. By contrast, Rpal_4085 demontrated significant electronic and structural differences from PioC. These differences likely explain its inability to reduce the LH-RC and highlight its distinct functional role. Overall, this work reveals the functional resilience of the pio operon pathway and further highlights the use of paramagnetic NMR for understanding key biological processes. [ABSTRACT FROM AUTHOR]

As sulfur is part of many essential protein cofactors such as iron–sulfur clusters, molybdenum cofactors, or lipoic acid, its mobilization from cysteine represents a fundamental process. The abstraction of the sulfur atom from cysteine is catalysed by highly conserved pyridoxal 5ʹ-phosphate-dependent enzymes called cysteine desulfurases. The desulfuration of cysteine leads to the formation of a persulfide group on a conserved catalytic cysteine and the concomitant release of alanine. Sulfur is then transferred from cysteine desulfurases to different targets. Numerous studies have focused on cysteine desulfurases as sulfur-extracting enzymes for iron–sulfur cluster synthesis in mitochondria and chloroplasts but also for molybdenum cofactor sulfuration in the cytosol. Despite this, knowledge about the involvement of cysteine desulfurases in other pathways is quite rudimentary, particularly in photosynthetic organisms. In this review, we summarize current understanding of the different groups of cysteine desulfurases and their characteristics in terms of primary sequence, protein domain architecture, and subcellular localization. In addition, we review the roles of cysteine desulfurases in different fundamental pathways and highlight the gaps in our knowledge to encourage future work on unresolved issues especially in photosynthetic organisms. [ABSTRACT FROM AUTHOR]

The mitochondrial electron transport chain (ETC) of apicomplexan parasites differs considerably from the ETC of the animals that these parasites infect, and is the target of numerous anti-parasitic drugs. The cytochrome c oxidase complex (Complex IV) of the apicomplexan Toxoplasma gondii ETC is more than twice the mass and contains subunits not found in human Complex IV, including a 13 kDa protein termed TgApiCox13. TgApiCox13 is homologous to a human iron-sulfur (Fe-S) cluster-containing protein called the mitochondrial inner NEET protein (HsMiNT) which is not a component of Complex IV in humans. Here, we establish that TgApiCox13 is a critical component of Complex IV in T. gondii, required for complex activity and stability. Furthermore, we demonstrate that TgApiCox13, like its human homolog, binds two Fe-S clusters. We show that the Fe-S clusters of TgApiCox13 are critical for ETC function, having an essential role in mediating Complex IV integrity. Our study provides the first functional characterisation of an Fe-S protein in Complex IV. Author summary: Complex IV is a canonical component of the electron transport chain (ETC) of eukaryotic mitochondria. Recent evidence indicates that considerable diversity exists in the protein composition of Complex IV in eukaryotes, although the functions of most novel Complex IV proteins remain a mystery. In this study, we characterize TgApiCox13, a novel Complex IV protein from the apicomplexan parasite Toxoplasma gondii. We demonstrate that TgApiCox13 is an iron-sulfur protein that is essential for parasite survival, functioning as a critical mediator of the activity and stability of Complex IV. We show that the Fe-S clusters of TgApiCox13 are critical for Complex IV function, playing a key role in mediating integrity of the complex. Our findings provide the first functional characterization of an Fe-S cluster protein in Complex IV, highlighting the diversity that exists in a core complex of the mitochondrial ETC in eukaryotes. [ABSTRACT FROM AUTHOR]