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A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in aging in Physcomitrella and Arabidopsis

ABSTRACT: Protein oxidation results from the reaction of amino-acid side chains with reactive oxygen species (ROS) and is mostly irreversible. In non-photosynthetic tissues, mitochondria are a main source of ROS, whereas plastids are the major source in photosynthetic tissues. Oxidized proteins suffer from decreased structural integrity and even loss of function, and their accumulation ultimately leads to cytotoxic aggregates. In mammals, this correlates with aging and is linked to several age-related pathologies. Mammalian proteolytic pathways for clearance of oxidized proteins are under intensive research, while mechanistic insights into this process in plants is scarce. AARE enzymes are ATP-independent serine proteases presumably acting on oxidized proteins and operating in a dual exo-/endopeptidase mode. They are found in all domains of life. Here, we investigated AARE enzymes in the moss Physcomitrella and the angiosperm Arabidopsis and identified three homologous nuclear genes in Physcomitrella (PpAARE1-3) and a single nuclear gene in Arabidopsis (AtAARE). Surprisingly, we observed triple localization of the proteins AtAARE and PpAARE1 to plastids, mitochondria and the cytosol in vivo, likely conserved across the plant lineage. This represents an ATP-independent possibility for degradation of oxidized proteins in the major source organelles of ROS in plants, which is distinct to mammals. Combinatorial knockout plants and protein interaction analysis revealed specific interactions of the moss AARE isoforms and functions in progressive aging. Analysis of an AtAARE T-DNA mutant further suggests conservation of AARE function in age-related development.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Physcomitrella Patens Subsp. Patens (moss)

TISSUE(S): Protonema

SUBMITTER: Ralf Reski

LAB HEAD: Ralf Reski

PROVIDER: PXD033854 | Pride | 2023-03-11

REPOSITORIES: Pride

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A deeply conserved protease, acylamino acid-releasing enzyme (AARE), acts in ageing in Physcomitrella and Arabidopsis.

Hoernstein Sebastian N W SNW Özdemir Buğra B van Gessel Nico N Miniera Alessandra A AA Rogalla von Bieberstein Bruno B Nilges Lars L Schweikert Farinha Joana J Komoll Ramona R Glauz Stella S Weckerle Tim T Scherzinger Friedrich F Rodriguez-Franco Marta M Müller-Schüssele Stefanie J SJ Reski Ralf R

Communications biology 20230117 1

Reactive oxygen species (ROS) are constant by-products of aerobic life. In excess, ROS lead to cytotoxic protein aggregates, which are a hallmark of ageing in animals and linked to age-related pathologies in humans. Acylamino acid-releasing enzymes (AARE) are bifunctional serine proteases, acting on oxidized proteins. AARE are found in all domains of life, albeit under different names, such as acylpeptide hydrolase (APEH/ACPH), acylaminoacyl peptidase (AAP), or oxidized protein hydrolase (OPH). ...[more]

PMID: 36650210

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Project description:As biopharmaceuticals, recombinant proteins have become indispensable tools in medicine. An increasing demand, not only in quantity but also in diversity, drives the constant development and improvement of production platforms. The N-glycosylation pattern on biopharmaceuticals plays important roles in activity, serum half-life and immunogenicity. Therefore, production platforms with tailored protein N-glycosylation are of great interest. Plant-based systems have already demonstrated their potential to produce pharmaceutically relevant recombinant proteins, although their N-glycan patterns differ from those in humans. Plants have shown great plasticity towards the manipulation of their glycosylation machinery, and some have already been glyco-engineered in order to avoid the attachment of plant-typical, putatively immunogenic sugar residues. This resulted in complex-type N-glycans with a core structure identical to the human one. Compared to humans, plants lack the ability to elongate these N-glycans with β1,4-linked galactoses and terminal sialic acids. However, these modifications, which require the activity of several mammalian enzymes, have already been achieved for Nicotiana benthamiana and the moss Physcomitrella. Here, we present the first step towards sialylation of recombinant glycoproteins in Physcomitrella, human β1,4-linked terminal N-glycan galactosylation, which was achieved by the introduction of a chimeric β1,4-galactosyltransferase (FTGT). This chimeric enzyme consists of the moss α1,4-fucosyltransferase transmembrane domain, fused to the catalytic domain of the human β1,4-galactosyltransferase. Stable FTGT expression led to the desired β1,4-galactosylation. However, additional pentoses of unknown identity were also observed. The nature of these pentoses was subsequently determined by Western blot and enzymatic digestion followed by mass spectrometric analysis and resulted in their identification as α-linked arabinoses. Since a pentosylation of β1,4-galactosylated N-glycans was reported earlier, e.g. on recombinant human erythropoietin produced in glyco-engineered Nicotiana tabacum, this phenomenon is of a more general importance for plant-based production platforms. Arabinoses, which are absent in humans, may prevent the full humanization of plant-derived products. Therefore, the identification of these pentoses as arabinoses is important as it creates the basis for their abolishment to ensure the production of safe biopharmaceuticals in plant-based systems.

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	Interaction_out_AARE1_CoIP.txt	Txt
	Interaction_out_AARE2_CoIP.txt	Txt
	QEplus031490.raw	Raw
	QEplus031491.raw	Raw
	QEplus031492.raw	Raw