Assisting the implementation of screening for type 1 diabetes by using artificial intelligence on publicly available data.

The type 1 diabetes community is coalescing around the benefits and advantages of early screening for disease risk. To be accepted by healthcare providers, regulatory authorities and payers, screening programmes need to show that the testing variables allow accurate risk prediction and that individualised risk-informed monitoring plans are established, as well as operational feasibility, cost-effectiveness and acceptance at population level. Artificial intelligence (AI) has the potential to contribute to solving these issues, starting with the identification and stratification of at-risk individuals. ASSET (AI for Sustainable Prevention of Autoimmunity in the Society; www.asset.healthcare ) is a public/private consortium that was established to contribute to research around screening for type 1 diabetes and particularly to how AI can drive the implementation of a precision medicine approach to disease prevention. ASSET will additionally focus on issues pertaining to operational implementation of screening. The authors of this article, researchers and clinicians active in the field of type 1 diabetes, met in an open forum to independently debate key issues around screening for type 1 diabetes and to advise ASSET. The potential use of AI in the analysis of longitudinal data from observational cohort studies to inform the design of improved, more individualised screening programmes was also discussed. A key issue was whether AI would allow the research community and industry to capitalise on large publicly available data repositories to design screening programmes that allow the early detection of individuals at high risk and enable clinical evaluation of preventive therapies. Overall, AI has the potential to revolutionise type 1 diabetes screening, in particular to help identify individuals who are at increased risk of disease and aid in the design of appropriate follow-up plans. We hope that this initiative will stimulate further research on this very timely topic.

Phase III, randomised, double-blind, placebo-controlled, multicentre trial to evaluate the efficacy and safety of rhGAD65 to preserve endogenous beta cell function in adolescents and adults with recently diagnosed type 1 diabetes, carrying the genetic HLA DR3-DQ2 haplotype: the DIAGNODE-3 study protocol.

INTRODUCTION: Type 1 diabetes (T1D) is an autoimmune disease leading to the destruction of the insulin-producing beta cells resulting in insulin deficiency and hyperglycaemic. Today, no approved therapy exists to halt this detrimental immunologic process. In a recent phase 2b study, intralymphatic administration of recombinant human glutamic acid decarboxylase 65 kDa (rhGAD65) adsorbed to Alhydrogel adjuvant to individuals recently diagnosed with T1D and carrying the HLA DR3-DQ2 haplotype showed promising results in preserving endogenous insulin secretion, confirming the results of a large meta-analysis of three randomised placebo-controlled trials of subcutaneous rhGAD65. The aim of the current precision medicine phase 3 study is to determine whether intralymphatic administration of rhGAD65 preserves insulin secretion and improves glycaemic control in presumed responder individuals with recently diagnosed T1D carrying HLA DR3-DQ2. METHODS AND ANALYSIS: Individuals ≥12 and <29 years recently diagnosed with T1D (<6 months) will be screened for the HLA DR3-DQ2 haplotype, endogenous insulin production estimated by fasting C-peptide and presence of GAD65 antibodies. 330 patients are planned to be randomised to 3 monthly intralymphatic injections of rhGAD65 or placebo (both accompanied by oral vitamin D supplementation), followed by 22 months of follow-up. The study is powered to detect a treatment effect in the two coprimary endpoints; change from baseline in AUC(0-120min) C-peptide levels during a mixed meal tolerance test, and change from baseline in glycaemic control estimated by haemoglobin A1c at 24 months. Secondary endpoints include effects on glucose patterns collected by masked continuous glucose monitoring, proportion of patients in partial remission and number of episodes of severe hypoglycaemia and/or diabetic ketoacidosis. ETHICS AND DISSEMINATION: The trial is approved by Ethics Committees in Poland (124/2021), the Netherlands (R21.089), Sweden (2021-05063), Czech Republic (EK-1144/21), Germany (2021361) and Spain (21/2021). Results will be published in international peer-reviewed scientific journals and presented at national and international conferences. TRIAL REGISTRATION NUMBER: EudraCT identifier: 2021-002731-32, NCT identifier: NCT05018585.

The dual-targeted prolyl aminopeptidase PAP1 is involved in proline accumulation in response to stress and during pollen development.

Plant endosymbiotic organelles such as mitochondria and chloroplasts harbour a wide array of biochemical reactions. As a part of protein homeostasis to maintain organellar activity and stability, unwanted proteins and peptides need to be completely degraded in a stepwise mechanism termed the processing pathway, where at the last stage single amino acids are released by aminopeptidases. Here, we determined the molecular and physiological functions of a prolyl aminopeptidase homologue PAP1 (At2g14260) that is able to release N-terminal proline. Transcript analyses demonstrate that an alternative transcription start site gives rise to two alternative transcripts, generating two in-frame proteins PAP1.1 and PAP1.2. Subcellular localization studies revealed that the longer isoform PAP1.1, which contains a 51 residue N-terminal extension, is exclusively targeted to chloroplasts, while the truncated isoform PAP1.2 is located in the cytosol. Distinct expression patterns in different tissues and developmental stages were observed. Investigations into the physiological role of PAP1 using loss-of-function mutants revealed that PAP1 activity may be involved in proline homeostasis and accumulation, required for pollen development and tolerance to osmotic stress. Enzymatic activity, subcellular location, and expression patterns of PAP1 suggest a role in the chloroplastic peptide processing pathway and proline homeostasis.

A mitochondrial prolyl aminopeptidase PAP2 releases N-terminal proline and regulates proline homeostasis during stress response.

Most mitochondrial proteins are synthesised in the cytosol and targeted into the organelle via N-terminal targeting peptides that are cleaved upon import. The free targeting peptide is subsequently processed in a stepwise manner, with single amino acids released as final products. Here, we have characterised a proline-cleaving aminopeptidase in Arabidopsis thaliana, prolyl aminopeptidase-2 (PAP2, At3g61540). Activity assays show that PAP2 has a preferred activity to hydrolyse N-terminal proline. Protein localisation studies revealed that PAP2 is exclusively targeted to mitochondria. Characterisation of pap2 mutants show defective pollen, enhanced dark-induced senescence and increased susceptibility to abiotic stresses, which are likely attributed to a reduced level of accumulated free proline. Taken together, these results demonstrate the role of PAP2 in proline cleavage from mitochondrial peptides and proline homeostasis, which is required for the development of male gametophyte, tolerance to abiotic stresses, and leaf senescence.

A Mitochondrial LYR Protein Is Required for Complex I Assembly.

Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.

Accumulation of endogenous peptides triggers a pathogen stress response in Arabidopsis thaliana.

The stepwise degradation of peptides to amino acids in plant mitochondria and chloroplasts is catalyzed by a network of oligopeptidases (presequence protease PreP, organellar oligopeptidase OOP) and aminopeptidases. In the present report, we show that the lack of oligopeptidase activity in Arabidopsis thaliana results in the accumulation of endogenous free peptides, mostly of chloroplastic origin (targeting peptides and degradation products). Using mRNA sequencing and deep coverage proteomics, allowing for the identification of 17 000 transcripts and 11 000 proteins, respectively, we uncover a peptide-stress response occurring in plants lacking PreP and OOP oligopeptidase activity. The peptide-stress response results in the activation of the classical plant defense pathways in the absence of pathogenic challenge. The constitutive activation of the pathogen-defense pathways imposes a strong growth penalty and a reduction of the plants reproductive fitness. Our results indicate that the absence of organellar oligopeptidases PreP1/2 and OOP results in the accumulation of peptides that are perceived as pathogenic effectors and activate the signaling pathways of plant-defense response.

Mechanism of Peptide Binding and Cleavage by the Human Mitochondrial Peptidase Neurolysin.

Proteolysis plays an important role in mitochondrial biogenesis, from the processing of newly imported precursor proteins to the degradation of mitochondrial targeting peptides. Disruption of peptide degradation activity in yeast, plant and mammalian mitochondria is known to have deleterious consequences for organism physiology, highlighting the important role of mitochondrial peptidases. In the present work, we show that the human mitochondrial peptidase neurolysin (hNLN) can degrade mitochondrial presequence peptides as well as other fragments up to 19 amino acids long. The crystal structure of hNLNE475Q in complex with the products of neurotensin cleavage at 2.7Å revealed a closed conformation with an internal cavity that restricts substrate length and highlighted the mechanism of enzyme opening/closing that is necessary for substrate binding and catalytic activity. Analysis of peptide degradation in vitro showed that hNLN cooperates with presequence protease (PreP or PITRM1) in the degradation of long targeting peptides and amyloid-ß peptide, Aß1-40, associated with Alzheimer disease, particularly cleaving the hydrophobic fragment Aß35-40. These findings suggest that a network of proteases may be required for complete degradation of peptides localized in mitochondria.

A multi-step peptidolytic cascade for amino acid recovery in chloroplasts.

Plastids (including chloroplasts) are subcellular sites for a plethora of proteolytic reactions, required in functions ranging from protein biogenesis to quality control. Here we show that peptides generated from pre-protein maturation within chloroplasts of Arabidopsis thaliana are degraded to amino acids by a multi-step peptidolytic cascade consisting of oligopeptidases and aminopeptidases, effectively allowing the recovery of single amino acids within these organelles.

Plant-Specific Preprotein and Amino Acid Transporter Proteins Are Required for tRNA Import into Mitochondria.

A variety of eukaryotes, in particular plants, do not contain the required number of tRNAs to support the translation of mitochondria-encoded genes and thus need to import tRNAs from the cytosol. This study identified two Arabidopsis (Arabidopsis thaliana) proteins, Tric1 and Tric2 (for tRNA import component), which on simultaneous inactivation by T-DNA insertion lines displayed a severely delayed and chlorotic growth phenotype and significantly reduced tRNA import capacity into isolated mitochondria. The predicted tRNA-binding domain of Tric1 and Tric2, a sterile-α-motif at the C-terminal end of the protein, was required to restore tRNA uptake ability in mitochondria of complemented plants. The purified predicted tRNA-binding domain binds the T-arm of the tRNA for alanine with conserved lysine residues required for binding. T-DNA inactivation of both Tric proteins further resulted in an increase in the in vitro rate of in organello protein synthesis, which was mediated by a reorganization of the nuclear transcriptome, in particular of genes encoding a variety of proteins required for mitochondrial gene expression at both the transcriptional and translational levels. The characterization of Tric1/2 provides mechanistic insight into the process of tRNA import into mitochondria and supports the theory that the tRNA import pathway resulted from the repurposing of a preexisting protein import apparatus.

Divergent evolution of the M3A family of metallopeptidases in plants.

Plants, as stationary organisms, have developed mechanisms allowing them efficient resource reallocation and a response to changing environmental conditions. One of these mechanisms is proteome remodeling via a broad peptidase network present in various cellular compartments including mitochondria and chloroplasts. The genome of the model plant Arabidopsis thaliana encodes as many as 616 putative peptidase-coding genes organized in 55 peptidase families. In this study, we describe the M3A family of peptidases, which comprises four members: mitochondrial and chloroplastic oligopeptidase (OOP), cytosolic oligopeptidase (CyOP), mitochondrial octapeptidyl aminopeptidase 1 (Oct1) and plant-specific protein of M3 family (PSPM3) of unknown function. We have analyzed the evolutionary conservation of M3A peptidases across plant species and the functional specialization of the three distinct subfamilies. We found that the subfamily-containing OOP and CyOP-like peptidases, responsible for oligopeptide degradation in the endosymbiotic organelles (OOP) or in the cytosol (CyOP), are highly conserved in all kingdoms of life. The Oct1-like peptidase subfamily involved in pre-protein maturation in mitochondria is conserved in all eukaryotes, whereas the PSPM3-like protein subfamily is strictly conserved in higher plants only and is of unknown function. Specific characteristics within PSPM3 sequences, i.e. occurrence of a N-terminal transmembrane domain and amino acid changes in distal substrate-binding motif, distinguish PSPM3 proteins from other members of M3A family. We performed peptidase activity measurements to analyze the role of substrate-binding residues in the different Arabidopsis M3A paralogs.

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function.

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

A flowchart to analyze protease activity in plant mitochondria.

Proteases are one of the most abundant classes of enzymes and are involved in a plethora of biological processes in many cellular compartments, including the mitochondria. To understand the role of proteases is essential to determine their substrate repertoire, preferably in an in vivo setting. In this chapter we describe general guidelines to analyze protease activity using several strategies, from in-gel analysis to mass spectrometry mapping of the cleavage site(s) and fluorogenic probes that can easily be used in vivo. To exemplify this flowchart, we used the recently characterized organellar oligopeptidase of Arabidopsis (Arabidopsis thaliana), an enzyme that takes part in degradation of short peptides within mitochondria and chloroplasts.

Protein import into plant mitochondria: signals, machinery, processing, and regulation.

The majority of more than 1000 proteins present in mitochondria are imported from nuclear-encoded, cytosolically synthesized precursor proteins. This impressive feat of transport and sorting is achieved by the combined action of targeting signals on mitochondrial proteins and the mitochondrial protein import apparatus. The mitochondrial protein import apparatus is composed of a number of multi-subunit protein complexes that recognize, translocate, and assemble mitochondrial proteins into functional complexes. While the core subunits involved in mitochondrial protein import are well conserved across wide phylogenetic gaps, the accessory subunits of these complexes differ in identity and/or function when plants are compared with Saccharomyces cerevisiae (yeast), the model system for mitochondrial protein import. These differences include distinct protein import receptors in plants, different mechanistic operation of the intermembrane protein import system, the location and activity of peptidases, the function of inner-membrane translocases in linking the outer and inner membrane, and the association/regulation of mitochondrial protein import complexes with components of the respiratory chain. Additionally, plant mitochondria share proteins with plastids, i.e. dual-targeted proteins. Also, the developmental and cell-specific nature of mitochondrial biogenesis is an aspect not observed in single-celled systems that is readily apparent in studies in plants. This means that plants provide a valuable model system to study the various regulatory processes associated with protein import and mitochondrial biogenesis.

Shredding the signal: targeting peptide degradation in mitochondria and chloroplasts.

The biogenesis and functionality of mitochondria and chloroplasts depend on the constant turnover of their proteins. The majority of mitochondrial and chloroplastic proteins are imported as precursors via their N-terminal targeting peptides. After import, the targeting peptides are cleaved off and degraded. Recent work has elucidated a pathway involved in the degradation of targeting peptides in mitochondria and chloroplasts, with two proteolytic components: the presequence protease (PreP) and the organellar oligopeptidase (OOP). PreP and OOP are specialized in degrading peptides of different lengths, with the substrate restriction being dictated by the structure of their proteolytic cavities. The importance of the intraorganellar peptide degradation is highlighted by the fact that elimination of both oligopeptidases affects growth and development of Arabidopsis thaliana.

The novel component Kgd4 recruits the E3 subunit to the mitochondrial α-ketoglutarate dehydrogenase.

The mitochondrial citric acid cycle is a central hub of cellular metabolism, providing intermediates for biosynthetic pathways and channeling electrons to the respiratory chain complexes. In this study, we elucidated the composition and organization of the multienzyme complex α-ketoglutarate dehydrogenase (α-KGDH). In addition to the three classical E1-E3 subunits, we identified a novel component, Kgd4 (Ymr31/MRPS36), which was previously assigned to be a subunit of the mitochondrial ribosome. Biochemical analyses demonstrate that this protein plays an evolutionarily conserved role in the organization of mitochondrial α-KGDH complexes of fungi and animals. By binding to both the E1-E2 core and the E3 subunit, Kgd4 acts as a molecular adaptor that is necessary to a form a stable α-KGDH enzyme complex. Our work thus reveals a novel subunit of a key citric acid-cycle enzyme and shows how this large complex is organized.

Phenotypical consequences of expressing the dually targeted Presequence Protease, AtPreP, exclusively in mitochondria.

Endosymbiotic organelles, mitochondria and chloroplasts, are sites of an intensive protein synthesis and degradation. A consequence of these processes is production of both free targeting peptides, i.e. mitochondrial presequences and chloroplastic transit peptides, and other short unstructured peptides. Mitochondrial, as well as chloroplastic peptides are degraded by Presequence Protease (PreP), which is dually targeted to mitochondrial matrix and chloroplastic stroma. Elimination of PreP in Arabidopsis thaliana leads to growth retardation, chlorosis and impairment of mitochondrial functions potentially due to the accumulation of targeting peptides. In this work we analyzed the influence of the restoration of mitochondrial peptide degradation by AtPreP on plant phenotype. We showed that exclusive mitochondrial expression of AtPreP results in total restoration of the proteolytic activity, but it does not restore the wild-type phenotype. The plants grow shorter roots and smaller rosettes compared to the plants expressing AtPreP1 in both mitochondria and chloroplasts. With this analysis we are aiming at understanding the physiological impact of the role of the dually targeted AtPreP in single type of destination organelle.

Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts.

Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 Å, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 Å(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.

The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro.

Ammonium assimilation is tightly regulated in nitrogen-fixing bacteria; the target of regulation is primarily the activity of the key enzyme glutamine synthetase that is regulated by reversible covalent modification by AMP groups in reactions catalysed by the bifunctional adenylyltransferase (ATase). The properties and regulation of ATase from Escherichia coli have been studied in great detail. We have investigated the regulation of ATase from Rhodospirillum rubrum, a photosynthetic nitrogen-fixing bacterium. In this diazotroph, nitrogenase is regulated at the metabolic level in addition to the transcriptional regulation operating in all diazotrophic bacteria, which makes understanding the regulatory features of nitrogen assimilation even more interesting. We show that in R. rubrum, in contrast to the E. coli system, ATase is primarily regulated by alpha-ketoglutarate and that glutamine has no effect on neither the adenylylation nor the deadenylylation of glutamine synthetase. Furthermore, the role of the regulatory P(II) proteins is only to stimulate the adenylylation reaction, as there is no effect on the reverse reaction. We propose that in R. rubrum and possibly other diazotrophs alpha-ketoglutarate plays the central role in the regulation of ATase and thus glutamine synthetase activity.