scholarly journals A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3

2020 ◽  
Vol 21 (21) ◽  
pp. 8121
Author(s):  
Nathalie Berger ◽  
Florence Vignols ◽  
Brigitte Touraine ◽  
Maël Taupin-Broggini ◽  
Valérie Rofidal ◽  
...  
Keyword(s):  
De Novo ◽  
Protein Localization ◽  
Expression Patterns ◽  
Specific Protein ◽  
Protein Accumulation ◽  
Loss Of Function ◽  
Iron Sulfur ◽  
Proteomic Approach ◽  
Client Proteins ◽  
S Proteins

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

scholarly journals Identification of client iron–sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana

2020 ◽  
Vol 71 (14) ◽  
pp. 4171-4187 ◽  
Author(s):  
Nathalie Berger ◽  
Florence Vignols ◽  
Jonathan Przybyla-Toscano ◽  
Mélanie Roland ◽  
Valérie Rofidal ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
De Novo ◽  
Bimolecular Fluorescence Complementation ◽  
In Planta ◽  
Chlorophyll Metabolism ◽  
Transfer Protein ◽  
Iron Sulfur ◽  
Bimolecular Fluorescence Complementation Assay ◽  
Client Proteins ◽  
S Proteins

Abstract Iron–sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

From the discovery to molecular understanding of cellular iron-sulfur protein biogenesis

2020 ◽  
Vol 401 (6-7) ◽  
pp. 855-876 ◽  
Author(s):  
Roland Lill
Keyword(s):  
De Novo ◽  
Current Knowledge ◽  
Protein Stabilization ◽  
S Protein ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
Sulfur Protein ◽  
Initial Discovery ◽  
S Proteins

AbstractProtein cofactors often are the business ends of proteins, and are either synthesized inside cells or are taken up from the nutrition. A cofactor that strictly needs to be synthesized by cells is the iron-sulfur (Fe/S) cluster. This evolutionary ancient compound performs numerous biochemical functions including electron transfer, catalysis, sulfur mobilization, regulation and protein stabilization. Since the discovery of eukaryotic Fe/S protein biogenesis two decades ago, more than 30 biogenesis factors have been identified in mitochondria and cytosol. They support the synthesis, trafficking and target-specific insertion of Fe/S clusters. In this review, I first summarize what led to the initial discovery of Fe/S protein biogenesis in yeast. I then discuss the function and localization of Fe/S proteins in (non-green) eukaryotes. The major part of the review provides a detailed synopsis of the three major steps of mitochondrial Fe/S protein biogenesis, i.e. the de novo synthesis of a [2Fe-2S] cluster on a scaffold protein, the Hsp70 chaperone-mediated transfer of the cluster and integration into [2Fe-2S] recipient apoproteins, and the reductive fusion of [2Fe-2S] to [4Fe-4S] clusters and their subsequent assembly into target apoproteins. Finally, I summarize the current knowledge of the mechanisms underlying the maturation of cytosolic and nuclear Fe/S proteins.

scholarly journals The Yeast Scaffold Proteins Isu1p and Isu2p Are Required inside Mitochondria for Maturation of Cytosolic Fe/S Proteins

2004 ◽  
Vol 24 (11) ◽  
pp. 4848-4857 ◽  
Author(s):  
Jana Gerber ◽  
Karina Neumann ◽  
Corinna Prohl ◽  
Ulrich Mühlenhoff ◽  
Roland Lill
Keyword(s):  
Iron Homeostasis ◽  
De Novo ◽  
Mitochondrial Targeting ◽  
Protein Maturation ◽  
Iron Sulfur Cluster ◽  
Subcellular Compartment ◽  
S Protein ◽  
Iron Sulfur ◽  
Protein Biogenesis ◽  
S Proteins

ABSTRACT Iron-sulfur (Fe/S) proteins are located in mitochondria, cytosol, and nucleus. Mitochondrial Fe/S proteins are matured by the iron-sulfur cluster (ISC) assembly machinery. Little is known about the formation of Fe/S proteins in the cytosol and nucleus. A function of mitochondria in cytosolic Fe/S protein maturation has been noted, but small amounts of some ISC components have been detected outside mitochondria. Here, we studied the highly conserved yeast proteins Isu1p and Isu2p, which provide a scaffold for Fe/S cluster synthesis. We asked whether the Isu proteins are needed for biosynthesis of cytosolic Fe/S clusters and in which subcellular compartment the Isu proteins are required. The Isu proteins were found to be essential for de novo biosynthesis of both mitochondrial and cytosolic Fe/S proteins. Several lines of evidence indicate that Isu1p and Isu2p have to be located inside mitochondria in order to perform their function in cytosolic Fe/S protein maturation. We were unable to mislocalize Isu1p to the cytosol due to the presence of multiple, independent mitochondrial targeting signals in this protein. Further, the bacterial homologue IscU and the human Isu proteins (partially) complemented the defects of yeast Isu protein-depleted cells in growth rate, Fe/S protein biogenesis, and iron homeostasis, yet only after targeting to mitochondria. Together, our data suggest that the Isu proteins need to be localized in mitochondria to fulfill their functional requirement in Fe/S protein maturation in the cytosol.

scholarly journals SOX2 Plays a Critical Role in the Pituitary, Forebrain, and Eye during Human Embryonic Development

2008 ◽  
Vol 93 (5) ◽  
pp. 1865-1873 ◽  
Author(s):  
Daniel Kelberman ◽  
Sandra C. P. de Castro ◽  
Shuwen Huang ◽  
John A. Crolla ◽  
Rodger Palmer ◽  
...  
Keyword(s):  
Embryonic Development ◽  
Anterior Pituitary ◽  
De Novo ◽  
Hypogonadotropic Hypogonadism ◽  
Expression Patterns ◽  
Critical Role ◽  
Loss Of Function ◽  
De Novo Mutations ◽  
Direct Role

Abstract Context: Heterozygous, de novo mutations in the transcription factor SOX2 are associated with bilateral anophthalmia or severe microphthalmia and hypopituitarism. Variable additional abnormalities include defects of the corpus callosum and hippocampus. Objective: We have ascertained a further three patients with severe eye defects and pituitary abnormalities who were screened for mutations in SOX2. To provide further evidence of a direct role for SOX2 in hypothalamo-pituitary development, we have studied the expression of the gene in human embryonic tissues. Results: All three patients harbored heterozygous SOX2 mutations: a deletion encompassing the entire gene, an intragenic deletion (c.70_89del), and a novel nonsense mutation (p.Q61X) within the DNA binding domain that results in impaired transactivation. We also show that human SOX2 can inhibit β-catenin-driven reporter gene expression in vitro, whereas mutant SOX2 proteins are unable to repress efficiently this activity. Furthermore, we show that SOX2 is expressed throughout the human brain, including the developing hypothalamus, as well as Rathke’s pouch, the developing anterior pituitary, and the eye. Conclusions: Patients with SOX2 mutations often manifest the unusual phenotype of hypogonadotropic hypogonadism, with sparing of other pituitary hormones despite anterior pituitary hypoplasia. SOX2 expression patterns in human embryonic development support a direct involvement of the protein during development of tissues affected in these individuals. Given the critical role of Wnt-signaling in the development of most of these tissues, our data suggest that a failure to repress the Wnt-β-catenin pathway could be one of the underlying pathogenic mechanisms associated with loss-of-function mutations in SOX2.

scholarly journals Varying intolerance of gene pathways to mutational classes explain genetic convergence across neuropsychiatric disorders

2016 ◽  
Author(s):  
Shahar Shohat ◽  
Eyal Ben-David ◽  
Sagiv Shifman
Keyword(s):  
Gene Expression ◽  
Genome Wide Association Study ◽  
De Novo ◽  
Expression Patterns ◽  
Neuropsychiatric Disorders ◽  
Autism Spectrum ◽  
Specific Gene ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Brain Gene Expression

AbstractGenetic susceptibility to Intellectual disability (ID), autism spectrum disorder (ASD) and schizophrenia (SCZ) often arises from mutations in the same genes, suggesting that they share common mechanisms. We studied genes with de novo mutations in the three disorders and genes implicated by SCZ genome-wide association study (GWAS). Using biological annotations and brain gene expression, we show that mutation class explains enrichment patterns more than specific disorder. Genes with loss of function mutations and genes with missense mutations were enriched with different pathways, shared with genes intolerant to mutations. Specific gene expression patterns were found for each disorder. ID genes were preferentially expressed in fetal cortex, ASD genes also in fetal cerebellum and striatum, and genes associated with SCZ were most significantly enriched in adolescent cortex. Our study suggests that convergence across neuropsychiatric disorders stems from vulnerable pathways to genetic variations, but spatiotemporal activity of genes contributes to specific phenotypes.

Mechanisms of Mitochondrial Iron-Sulfur Protein Biogenesis

2020 ◽  
Vol 89 (1) ◽  
pp. 471-499 ◽  
Author(s):  
Roland Lill ◽  
Sven-A. Freibert
Keyword(s):  
Molecular Mechanisms ◽  
De Novo ◽  
Lipid Synthesis ◽  
Cluster Assembly ◽  
Iron Sulfur Cluster ◽  
Atp Production ◽  
Cellular Iron ◽  
Iron Sulfur ◽  
Sulfur Protein ◽  
S Proteins

Mitochondria are essential in most eukaryotes and are involved in numerous biological functions including ATP production, cofactor biosyntheses, apoptosis, lipid synthesis, and steroid metabolism. Work over the past two decades has uncovered the biogenesis of cellular iron-sulfur (Fe/S) proteins as the essential and minimal function of mitochondria. This process is catalyzed by the bacteria-derived iron-sulfur cluster assembly (ISC) machinery and has been dissected into three major steps: de novo synthesis of a [2Fe-2S] cluster on a scaffold protein; Hsp70 chaperone–mediated trafficking of the cluster and insertion into [2Fe-2S] target apoproteins; and catalytic conversion of the [2Fe-2S] into a [4Fe-4S] cluster and subsequent insertion into recipient apoproteins. ISC components of the first two steps are also required for biogenesis of numerous essential cytosolic and nuclear Fe/S proteins, explaining the essentiality of mitochondria. This review summarizes the molecular mechanisms underlying the ISC protein–mediated maturation of mitochondrial Fe/S proteins and the importance for human disease.

scholarly journals Proteomic Analysis of KCNK3 Loss of Expression Identified Dysregulated Pathways in Pulmonary Vascular Cells

2020 ◽  
Vol 21 (19) ◽  
pp. 7400 ◽  
Author(s):  
Hélène Le Ribeuz ◽  
Florent Dumont ◽  
Guillaume Ruellou ◽  
Mélanie Lambert ◽  
Thierry Balliau ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Heme Oxygenase 1 ◽  
Loss Of Function ◽  
Apoptosis Resistance ◽  
Purine Nucleotides ◽  
Proteomic Approach ◽  
Pulmonary Arterial ◽  
And Migration

The physiopathology of pulmonary arterial hypertension (PAH) is characterized by pulmonary artery smooth muscle cell (PASMC) and endothelial cell (PAEC) dysfunction, contributing to pulmonary arterial obstruction and PAH progression. KCNK3 loss of function mutations are responsible for the first channelopathy identified in PAH. Loss of KCNK3 function/expression is a hallmark of PAH. However, the molecular mechanisms involved in KCNK3 dysfunction are mostly unknown. To identify the pathological molecular mechanisms downstream of KCNK3 in human PASMCs (hPASMCs) and human PAECs (hPAECs), we used a Liquid Chromatography-Tandem Mass Spectrometry-based proteomic approach to identify the molecular pathways regulated by KCNK3. KCNK3 loss of expression was induced in control hPASMCs or hPAECs by specific siRNA targeting KCNK3. We found that the loss of KCNK3 expression in hPAECs and hPASMCs leads to 326 and 222 proteins differentially expressed, respectively. Among them, 53 proteins were common to hPAECs and hPASMCs. The specific proteome remodeling in hPAECs in absence of KCNK3 was mostly related to the activation of glycolysis, the superpathway of methionine degradation, and the mTOR signaling pathways, and to a reduction in EIF2 signaling pathways. In hPASMCs, we found an activation of the PI3K/AKT signaling pathways and a reduction in EIF2 signaling and the Purine Nucleotides De Novo Biosynthesis II and IL-8 signaling pathways. Common to hPAECs and hPASMCs, we found that the loss of KCNK3 expression leads to the activation of the NRF2-mediated oxidative stress response and a reduction in the interferon pathway. In the hPAECs and hPASMCs, we found an increased expression of HO-1 (heme oxygenase-1) and a decreased IFIT3 (interferon-induced proteins with tetratricopeptide repeats 3) (confirmed by Western blotting), allowing us to identify these axes to understand the consequences of KCNK3 dysfunction. Our experiments, based on the loss of KCNK3 expression by a specific siRNA strategy in control hPAECs and hPASMCs, allow us to identify differences in the activation of several signaling pathways, indicating the key role played by KCNK3 dysfunction in the development of PAH. Altogether, these results allow us to better understand the consequences of KCNK3 dysfunction and suggest that KCNK3 loss of expression acts in favor of the proliferation and migration of hPASMCs and promotes the metabolic shift and apoptosis resistance of hPAECs.

scholarly journals miR156-targeted SPL10 controls Arabidopsis root meristem activity and root-derived de novo shoot regeneration via cytokinin responses

2019 ◽  
Vol 71 (3) ◽  
pp. 934-950 ◽  
Author(s):  
Carlos Hernán Barrera-Rojas ◽  
Gabriel Henrique Braga Rocha ◽  
Laura Polverari ◽  
Diego Armando Pinheiro Brito ◽  
Diego Silva Batista ◽  
...  
Keyword(s):  
Shoot Regeneration ◽  
Root Meristem ◽  
De Novo ◽  
Expression Patterns ◽  
Primary Root ◽  
Loss Of Function ◽  
Arabidopsis Root ◽  
Squamosa Promoter Binding Protein ◽  
Spl Genes ◽  
Dependent Pathway

Abstract Root growth is modulated by different factors, including phytohormones, transcription factors, and microRNAs (miRNAs). MicroRNA156 and its targets, the SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, define an age-dependent pathway that controls several developmental processes, including lateral root emergence. However, it remains unclear whether miR156-regulated SPLs control root meristem activity and root-derived de novo shoot regeneration. Here, we show that MIR156 and SPL genes have opposing expression patterns during the progression of primary root (PR) growth in Arabidopsis, suggesting that age cues may modulate root development. Plants with high miR156 levels display reduced meristem size, resulting in shorter primary root (PRs). Conversely, plants with reduced miR156 levels show higher meristem activity. Importantly, loss of function of SPL10 decreases meristem activity, while SPL10 de-repression increases it. Meristem activity is regulated by SPL10 probably through the reduction of cytokinin responses, via the modulation of type-B ARABIDOPSIS RESPONSE REGULATOR1(ARR1) expression. We also show that SPL10 de-repression in the PRs abolishes de novo shoot regenerative capacity by attenuating cytokinin responses. Our results reveal a cooperative regulation of root meristem activity and root-derived de novo shoot regeneration by integrating age cues with cytokinin responses via miR156-targeted SPL10.

scholarly journals Structure of the human frataxin-bound iron-sulfur cluster assembly complex provides insight into its activation mechanism

2019 ◽  
Author(s):  
Nicholas G. Fox ◽  
Xiaodi Yu ◽  
Xidong Feng ◽  
Henry J. Bailey ◽  
Alain Martelli ◽  
...  
Keyword(s):  
De Novo ◽  
Neurodegenerative Disorder ◽  
Acyl Carrier Protein ◽  
Local Environment ◽  
Zinc Ion ◽  
Activation Mechanism ◽  
Cluster Assembly ◽  
Loss Of Function ◽  
Iron Sulfur ◽  
Zinc Inhibition

AbstractIron-sulfur clusters (ISC) are essential in all life forms and carry out many crucial cellular functions. The core machinery for de novo ISC biosynthesis, located in the mitochondria matrix, is a five-protein complex containing the cysteine desulfurase NFS1 that is activated by frataxin (FXN), scaffold protein ISCU, accessory protein ISD11, and acyl-carrier protein ACP. Deficiency in FXN leads to the loss-of-function neurodegenerative disorder Friedreich’s ataxia (FRDA). Recently crystal structures depicting the inactive 3- and 4-way sub-complexes of the ISC biosynthesis machinery, lacking the key activator FXN, have been determined. Here, the 3.2 Å resolution cryo-electron microscopy structure of the FXN-bound active human complex, containing two copies of the NFS1-ISD11-ACP-ISCU-FXN hetero-pentamer, delineates for the first time in any organism the interactions of FXN with the component proteins. FXN binds at the interface of two NFS1 and one ISCU subunits, modifying the local environment of a bound zinc ion that would otherwise inhibit NFS1 activity in complexes without FXN. Our structure sheds light on how FXN facilitates ISC production through unlocking the zinc inhibition and stabilizing key loop conformations of NFS1 and ISCU at the protein-protein interfaces, and offers an explanation of how FRDA clinical mutations affect complex formation and FXN activation.

scholarly journals Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

2020 ◽  
Author(s):  
Jasmin Morandell ◽  
Lena A. Schwarz ◽  
Bernadette Basilico ◽  
Saren Tasciyan ◽  
Armel Nicolas ◽  
...  
Keyword(s):  
Neuronal Migration ◽  
Motor Coordination ◽  
De Novo ◽  
Critical Role ◽  
Autism Spectrum ◽  
Cytoskeletal Proteins ◽  
Inhibitory Neurons ◽  
Loss Of Function ◽  
Proteomic Approach

AbstractDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). Here, we used Cul3 mouse models to evaluate the consequences of Cul3 mutations in vivo. Our results show that Cul3 haploinsufficient mice exhibit deficits in motor coordination as well as ASD-relevant social and cognitive impairments. Cul3 mutant brain displays cortical lamination abnormalities due to defective neuronal migration and reduced numbers of excitatory and inhibitory neurons. In line with the observed abnormal columnar organization, Cul3 haploinsufficiency is associated with decreased spontaneous excitatory and inhibitory activity in the cortex. At the molecular level, employing a quantitative proteomic approach, we show that Cul3 regulates cytoskeletal and adhesion protein abundance in mouse embryos. Abnormal regulation of cytoskeletal proteins in Cul3 mutant neuronal cells results in atypical organization of the actin mesh at the cell leading edge, likely causing the observed migration deficits. In contrast to these important functions early in development, Cul3 deficiency appears less relevant at adult stages. In fact, induction of Cul3 haploinsufficiency in adult mice does not result in the behavioral defects observed in constitutive Cul3 haploinsufficient animals. Taken together, our data indicate that Cul3 has a critical role in the regulation of cytoskeletal proteins and neuronal migration and that ASD-associated defects and behavioral abnormalities are primarily due to Cul3 functions at early developmental stages.

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