Acquisition of hypoxia inducibility by oxygen sensing N-terminal cysteine oxidase in spermatophytes
N-terminal cysteine oxidases (NCOs) are enzymes that use molecular oxygen to oxidize the amino-terminal cysteine of specific proteins, thereby initiating the proteolytic N-degron pathway and thus conferring them oxygen-dependent instability. To expand the characterization of the plant family of NCOs (PCOs), we performed a phylogenetic analysis across different plant taxa in terms of sequence similarity and transcriptional regulation. Based on this survey, we propose a distinction of PCOs into two main groups: A-type and B-type sequences. A-type PCOs are conserved across all plant species and are generally unaffected at the mRNA level by oxygen availability. Instead, B-type PCOs differentiated in spermatophytes to acquire specific amino acid features and transcriptional regulation in response to hypoxia. Both groups of PCO proteins possess the ability to destabilize Cys-initiating proteins. Indeed, the inactivation of two A-type PCOs in Arabidopsis thaliana, PCO4 and PCO5, is sufficient to activate, at least partially, the anaerobic response in young seedlings, whereas the additional removal of B-type PCOs leads to a stronger induction of anaerobic genes and impairs plant growth and development. Our results show that both PCO types are required to regulate the anaerobic response in angiosperm. Therefore, while it is possible to distinguish two clades within the PCO family, separated by both amino acid features and transcriptional regulation, we conclude that they both contribute to restrain the anaerobic transcriptional program in normoxic conditions and together generate a molecular switch to toggle the hypoxic response in Arabidopsis.
Conserved N-terminal cysteine dioxygenases transduce responses to hypoxia in animals and plants
[a collaborative effort with Emily Flashman (Chemistry Department) and Sir. Peter Ratcliffe (Nuffield Department of Medicine)]
Organisms must respond to hypoxia to preserve oxygen homeostasis. We identify a thiol oxidase, previously assigned as cysteamine (2-aminoethanethiol) dioxygenase (ADO), as a low oxygen affinity (high-KmO2) amino-terminal cysteine dioxygenase that transduces the oxygen-regulated stability of proteins by the N-degron pathway in human cells. ADO catalyzes the conversion of amino-terminal cysteine to cysteine sulfinic acid and is related to the plant cysteine oxidases that mediate responses to hypoxia by an identical posttranslational modification. We show in human cells that ADO regulates RGS4/5 (regulator of G protein signaling) N-degron substrates, modulates G protein–coupled calcium ion signals and mitogen-activated protein kinase activity, and that its activity extends to other N-cysteine proteins including the angiogenic cytokine interleukin-32. Identification of a conserved enzymatic oxygen sensor in multicellular eukaryotes opens routes to better understanding and therapeutic targeting of adaptive responses to hypoxia.
Conservation of ethanol fermentation and its regulation in land plants
Ethanol fermentation is considered as one of the main metabolic adaptations to ensure energy production in higher plants under anaerobic conditions. Following this pathway, pyruvate is decarboxylated and reduced to ethanol with the concomitant oxidation of NADH to NAD+. Despite its acknowledgement as an essential metabolic strategy, the conservation of this pathway and its regulation throughout plant evolution have not been assessed so far. To address this question, we compared ethanol fermentation in species representing subsequent steps in plant evolution and related it to the structural features and transcriptional regulation of the two enzymes involved: pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). We observed that, despite the conserved ability to produce ethanol upon hypoxia in distant phyla, transcriptional regulation of the enzymes involved is not conserved in ancient plant lineages, whose ADH homologues do not share structural features distinctive for acetaldehyde/ethanol-processing enzymes. Moreover, Arabidopsis mutants devoid of ADH expression exhibited enhanced PDC activity and retained substantial ethanol production under hypoxic conditions. Therefore, we concluded that, whereas ethanol production is a highly conserved adaptation to low oxygen, its catalysis and regulation in land plants probably involve components that will be identified in the future.