CHEK2 germline variants identified in familial nonmedullary thyroid cancer lead to impaired protein structure and function.

Approximately 5 to 15% of nonmedullary thyroid cancers (NMTC) present in a familial form (familial nonmedullary thyroid cancers [FNMTC]). The genetic basis of FNMTC remains largely unknown, representing a limitation for diagnostic and clinical management. Recently, germline mutations in DNA repair-related genes have been described in cases with thyroid cancer (TC), suggesting a role in FNMTC etiology. Here, two FNMTC families were studied, each with two members affected with TC. Ninety-four hereditary cancer predisposition genes were analyzed through next-generation sequencing, revealing two germline CHEK2 missense variants (c.962A > C, p.E321A and c.470T > C, p.I157T), which segregated with TC in each FNMTC family. p.E321A, located in the CHK2 protein kinase domain, is a rare variant, previously unreported in the literature. Conversely, p.I157T, located in CHK2 forkhead-associated domain, has been extensively described, having conflicting interpretations of pathogenicity. CHK2 proteins (WT and variants) were characterized using biophysical methods, molecular dynamics simulations, and immunohistochemistry. Overall, biophysical characterization of these CHK2 variants showed that they have compromised structural and conformational stability and impaired kinase activity, compared to the WT protein. CHK2 appears to aggregate into amyloid-like fibrils in vitro, which opens future perspectives toward positioning CHK2 in cancer pathophysiology. CHK2 variants exhibited higher propensity for this conformational change, also displaying higher expression in thyroid tumors. The present findings support the utility of complementary biophysical and in silico approaches toward understanding the impact of genetic variants in protein structure and function, improving the current knowledge on CHEK2 variants' role in FNMTC genetic basis, with prospective clinical translation.

Microbial production of the plant flavanone hesperetin from caffeic acid.

OBJECTIVE: Hesperetin is an important O-methylated flavonoid produced by citrus fruits and of potential pharmaceutical relevance. The microbial biosynthesis of hesperetin could be a viable alternative to plant extraction, as plant extracts often yield complex mixtures of different flavonoids making it challenging to isolate pure compounds. In this study, hesperetin was produced from caffeic acid in the microbial host Escherichia coli. We combined a previously optimised pathway for the biosynthesis of the intermediate flavanone eriodictyol with a combinatorial library of plasmids expressing three candidate flavonoid O-methyltransferases. Moreover, we endeavoured to improve the position specificity of CCoAOMT7, a flavonoid O-methyltransferase from Arabidopsis thaliana that has been demonstrated to O-methylate eriodictyol in both the para- and meta-position, thus leading to a mixture of hesperetin and homoeriodictyol. RESULTS: The best performing flavonoid O-methyltransferase in our screen was found to be CCoAOMT7, which could produce up to 14.6 mg/L hesperetin and 3.8 mg/L homoeriodictyol from 3 mM caffeic acid in E. coli 5-alpha. Using a platform for enzyme engineering that scans the mutational space of selected key positions, predicting their structures using homology modelling and inferring their potential catalytic improvement using docking simulations, we were able to identify a CCoAOMT7 mutant with a two-fold higher position specificity for hesperetin. The mutant's catalytic activity, however, was considerably diminished. Our findings suggest that hesperetin can be created from central carbon metabolism in E. coli following the introduction of a caffeic acid biosynthesis pathway.

SARS-CoV-2 variants impact RBD conformational dynamics and ACE2 accessibility.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has killed over 6 million people and is having a devastating social and economic impact around the world. The rise of new variants of concern (VOCs) represents a difficult challenge due to the loss of vaccine and natural immunity, as well as increased transmissibility. All VOCs contain mutations in the spike glycoprotein, which mediates fusion between the viral and host cell membranes. The spike glycoprotein binds to angiotensin-converting enzyme 2 (ACE2) via its receptor binding domain (RBD) initiating the infection process. Attempting to understand the effect of RBD mutations in VOCs, a lot of attention has been given to the RBD-ACE2 interaction. However, this type of analysis ignores more indirect effects, such as the conformational dynamics of the RBD itself. Observing that some mutations occur in residues that are not in direct contact with ACE2, we hypothesized that they could affect the RBD conformational dynamics. To test this, we performed long atomistic (AA) molecular dynamics (MD) simulations to investigate the structural dynamics of wt RBD, and that of four VOCs (Alpha, Beta, Delta, and Omicron). Our results show that the wt RBD presents two distinct conformations: an "open" conformation where it is free to bind ACE2; and a "closed" conformation, where the RBM ridge blocks the binding surface. The Alpha and Beta variants shift the open/closed equilibrium towards the open conformation by roughly 20%, likely increasing ACE2 binding affinity. Simulations of the Delta and Omicron variants showed extreme results, with the closed conformation being rarely observed. The Delta variant also differed substantially from the other variants, alternating between the open conformation and an alternative "reversed" one, with a significantly changed orientation of the RBM ridge. This alternate conformation could provide a fitness advantage due to increased availability for ACE2 binding, and by aiding antibody escape through epitope occlusion. These results support the hypothesis that VOCs, and particularly the Omicron and Delta variants, impact RBD conformational dynamics in a direction that promotes efficient binding to ACE2 and, in the case of Delta, may assist antibody escape.

Parainfluenza Fusion Peptide Promotes Membrane Fusion by Assembling into Oligomeric Porelike Structures.

Paramyxoviruses are enveloped viruses harboring a negative-sense RNA genome that must enter the host's cells to replicate. In the case of the parainfluenza virus, the cell entry process starts with the recognition and attachment to target receptors, followed by proteolytic cleavage of the fusion glycoprotein (F) protein, exposing the fusion peptide (FP) region. The FP is responsible for binding to the target membrane, and it is believed to play a crucial role in the fusion process, but the mechanism by which the parainfluenza FP (PIFP) promotes membrane fusion is still unclear. To elucidate this matter, we performed biophysical experimentation of the PIFP in membranes, together with coarse grain (CG) and atomistic (AA) molecular dynamics (MD) simulations. The simulation results led to the pinpointing of the most important PIFP amino acid residues for membrane fusion and show that, at high concentrations, the peptide induces the formation of a water-permeable porelike structure. This structure promotes lipid head intrusion and lipid tail protrusion, which facilitates membrane fusion. Biophysical experimental results validate these findings, showing that, depending on the peptide/lipid ratio, the PIFP can promote fusion and/or membrane leakage. Our work furthers the understanding of the PIFP-induced membrane fusion process, which might help foster development in the field of viral entry inhibition.

The Importance of Lipid Conjugation on Anti-Fusion Peptides against Nipah Virus.

Nipah virus (NiV) is a recently emerging zoonotic virus that belongs to the Paramyxoviridae family and the Henipavirus genus. It causes a range of conditions, from asymptomatic infection to acute respiratory illness and fatal encephalitis. The high mortality rate of 40 to 90% ranks these viruses among the deadliest viruses known to infect humans. Currently, there is no antiviral drug available for Nipah virus disease and treatment is only supportive. Thus, there is an urgent demand for efficient antiviral therapies. NiV F protein, which catalyzes fusion between the viral and host membranes, is a potential target for antiviral drugs, as it is a key protein in the initial stages of infection. Fusion inhibitor peptides derived from the HRC-domain of the F protein are known to bind to their complementary domain in the protein's transient intermediate state, preventing the formation of a six-helix bundle (6HB) thought to be responsible for driving the fusion of the viral and cell membranes. Here, we evaluated the biophysical and structural properties of four different C-terminal lipid-tagged peptides. Different compositions of the lipid tags were tested to search for properties that might promote efficacy and broad-spectrum activity. Fluorescence spectroscopy was used to study the interaction of the peptides with biomembrane model systems and human blood cells. In order to understand the structural properties of the peptides, circular dichroism measurements and molecular dynamics simulations were performed. Our results indicate a peptide preference for cholesterol-enriched membranes and a lipid conjugation-driven stabilization of the peptide α-helical secondary structure. This work may contribute for the development of highly effective viral fusion against NiV inhibitors.

The nsp15 Nuclease as a Good Target to Combat SARS-CoV-2: Mechanism of Action and Its Inactivation with FDA-Approved Drugs.

The pandemic caused by SARS-CoV-2 is not over yet, despite all the efforts from the scientific community. Vaccination is a crucial weapon to fight this virus; however, we still urge the development of antivirals to reduce the severity and progression of the COVID-19 disease. For that, a deep understanding of the mechanisms involved in viral replication is necessary. nsp15 is an endoribonuclease critical for the degradation of viral polyuridine sequences that activate host immune sensors. This enzyme is known as one of the major interferon antagonists from SARS-CoV-2. In this work, a biochemical characterization of SARS-CoV-2 nsp15 was performed. We saw that nsp15 is active as a hexamer, and zinc can block its activity. The role of conserved residues from SARS-CoV-2 nsp15 was investigated, and N164 was found to be important for protein hexamerization and to contribute to the specificity to degrade uridines. Several chemical groups that impact the activity of this ribonuclease were also identified. Additionally, FDA-approved drugs with the capacity to inhibit the in vitro activity of nsp15 are reported in this work. This study is of utmost importance by adding highly valuable information that can be used for the development and rational design of therapeutic strategies.

Molecular mechanisms of the influenza fusion peptide: insights from experimental and simulation studies.

A key step in infections by enveloped viruses, such as influenza, is the fusion between the viral envelope and the host cell membrane, which allows the virus to insert its genetic material into the host cell and replicate. The influenza virus fusion process is promoted by hemagglutinin (HA), a glycoprotein that contains three identical monomers composed of two polypeptide chains (HA1 and HA2). Early studies on this protein revealed that HA-mediated fusion involves the insertion of the HA2 N-terminal segment into the host membrane and that this segment, known as the fusion peptide, is a key player in the fusion process. This mini-review highlights the main findings that have been obtained by experimental and computational studies on the HA fusion peptide, which give us a glimpse of its mode of action.

Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies.

Understanding SARS-CoV-2 evolution and host immunity is critical to control COVID-19 pandemics. At the core is an arms-race between SARS-CoV-2 antibody and angiotensin-converting enzyme 2 (ACE2) recognition, a function of the viral protein spike. Mutations in spike impacting antibody and/or ACE2 binding are appearing worldwide, imposing the need to monitor SARS-CoV2 evolution and dynamics in the population. Determining signatures in SARS-CoV-2 that render the virus resistant to neutralizing antibodies is critical. We engineered 25 spike-pseudotyped lentiviruses containing individual and combined mutations in the spike protein, including all defining mutations in the variants of concern, to identify the effect of single and synergic amino acid substitutions in promoting immune escape. We confirmed that E484K evades antibody neutralization elicited by infection or vaccination, a capacity augmented when complemented by K417N and N501Y mutations. In silico analysis provided an explanation for E484K immune evasion. E484 frequently engages in interactions with antibodies but not with ACE2. Importantly, we identified a novel amino acid of concern, S494, which shares a similar pattern. Using the already circulating mutation S494P, we found that it reduces antibody neutralization of convalescent and post-immunization sera, particularly when combined with E484K and with mutations able to increase binding to ACE2, such as N501Y. Our analysis of synergic mutations provides a signature for hotspots for immune evasion and for targets of therapies, vaccines and diagnostics.

New targets for drug design: importance of nsp14/nsp10 complex formation for the 3'-5' exoribonucleolytic activity on SARS-CoV-2.

SARS-CoV-2 virus has triggered a global pandemic with devastating consequences. The understanding of fundamental aspects of this virus is of extreme importance. In this work, we studied the viral ribonuclease nsp14, one of the most interferon antagonists from SARS-CoV-2. Nsp14 is a multifunctional protein with two distinct activities, an N-terminal 3'-to-5' exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), both critical for coronaviruses life cycle, indicating nsp14 as a prominent target for the development of antiviral drugs. In coronaviruses, nsp14 ExoN activity is stimulated through the interaction with the nsp10 protein. We have performed a biochemical characterization of nsp14-nsp10 complex from SARS-CoV-2. We confirm the 3'-5' exoribonuclease and MTase activities of nsp14 and the critical role of nsp10 in upregulating the nsp14 ExoN activity. Furthermore, we demonstrate that SARS-CoV-2 nsp14 N7-MTase activity is functionally independent of the ExoN activity and nsp10. A model from SARS-CoV-2 nsp14-nsp10 complex allowed mapping key nsp10 residues involved in this interaction. Our results show that a stable interaction between nsp10 and nsp14 is required for the nsp14-mediated ExoN activity of SARS-CoV-2. We studied the role of conserved DEDD catalytic residues of SARS-CoV-2 nsp14 ExoN. Our results show that motif I of ExoN domain is essential for the nsp14 function, contrasting to the functionality of these residues in other coronaviruses, which can have important implications regarding the specific pathogenesis of SARS-CoV-2. This work unraveled a basis for discovering inhibitors targeting specific amino acids in order to disrupt the assembly of this complex and interfere with coronaviruses replication.

Crossing the Wall: Characterization of the Multiheme Cytochromes Involved in the Extracellular Electron Transfer Pathway of Thermincola ferriacetica.

Bioelectrochemical systems (BES) are emerging as a suite of versatile sustainable technologies to produce electricity and added-value compounds from renewable and carbon-neutral sources using electroactive organisms. The incomplete knowledge on the molecular processes that allow electroactive organisms to exchange electrons with electrodes has prevented their real-world implementation. In this manuscript we investigate the extracellular electron transfer processes performed by the thermophilic Gram-positive bacteria belonging to the Thermincola genus, which were found to produce higher levels of current and tolerate higher temperatures in BES than mesophilic Gram-negative bacteria. In our study, three multiheme c-type cytochromes, Tfer_0070, Tfer_0075, and Tfer_1887, proposed to be involved in the extracellular electron transfer pathway of T. ferriacetica, were cloned and over-expressed in E. coli. Tfer_0070 (ImdcA) and Tfer_1887 (PdcA) were purified and biochemically characterized. The electrochemical characterization of these proteins supports a pathway of extracellular electron transfer via these two proteins. By contrast, Tfer_0075 (CwcA) could not be stabilized in solution, in agreement with its proposed insertion in the peptidoglycan wall. However, based on the homology with the outer-membrane cytochrome OmcS, a structural model for CwcA was developed, providing a molecular perspective into the mechanisms of electron transfer across the peptidoglycan layer in Thermincola.

Structural and functional impact of clinically relevant E1α variants causing pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate to acetyl-coenzyme A, hinging glycolysis and the tricarboxylic acid cycle. PDC deficiency, an inborn error of metabolism, has a broad phenotypic spectrum. Symptoms range from fatal lactic acidosis or progressive neuromuscular impairment in the neonatal period, to chronic neurodegeneration. Most disease-causing mutations in PDC deficiency affect the PDHA1 gene, encoding the α subunit of the PDC-E1 component. Detailed biophysical analysis of pathogenic protein variants is a challenging approach to support the design of therapies based on improving and correcting protein structure and function. Herein, we report the characterization of clinically relevant PDC-E1α variants identified in Portuguese PDC deficient patients. These variants bear amino acid substitutions in different structural regions of PDC-E1α. The structural and functional analyses of recombinant heterotetrameric (αα'ßß') PDC-E1 variants, combined with molecular dynamics (MD) simulations, show a limited impact of the amino acid changes on the conformational stability, apart from the increased propensity for aggregation of the p.R253G variant as compared to wild-type PDC-E1. However, all variants presented a functional impairment in terms of lower residual PDC-E1 enzymatic activity and ≈3-100 × lower affinity for the thiamine pyrophosphate (TPP) cofactor, in comparison with wild-type PDC-E1. MD simulations neatly showed generally decreased stability (increased flexibility) of all variants with respect to the WT heterotetramer, particularly in the TPP binding region. These results are discussed in light of disease severity of the patients bearing such mutations and highlight the difficulty of developing chaperone-based therapies for PDC deficiency.

ViralFP: A Web Application of Viral Fusion Proteins.

Viral fusion proteins are attached to the membrane of enveloped viruses (a group that includes Coronaviruses, Dengue, HIV and Influenza) and catalyze fusion between the viral and host membranes, enabling the virus to insert its genetic material into the host cell. Given the importance of these biomolecules, this work presents a centralized database containing the most relevant information on viral fusion proteins, available through a free-to-use web server accessible through the URL https://viralfp.bio.di.uminho.pt/. This web application contains several bioinformatic tools, such as Clustal sequence alignment and Weblogo, including as well a machine learning-based tool capable of predicting the location of fusion peptides (the component of fusion proteins that inserts into the host's cell membrane) within the fusion protein sequence. Given the crucial role of these proteins in viral infection, their importance as natural targets of our immune system and their potential as therapeutic targets, this web application aims to foster our ability to fight pathogenic viruses.

Effect of pH on the influenza fusion peptide properties unveiled by constant-pH molecular dynamics simulations combined with experiment.

The influenza virus fusion process, whereby the virus fuses its envelope with the host endosome membrane to release the genetic material, takes place in the acidic late endosome environment. Acidification triggers a large conformational change in the fusion protein, hemagglutinin (HA), which enables the insertion of the N-terminal region of the HA2 subunit, known as the fusion peptide, into the membrane of the host endosome. However, the mechanism by which pH modulates the molecular properties of the fusion peptide remains unclear. To answer this question, we performed the first constant-pH molecular dynamics simulations of the influenza fusion peptide in a membrane, extending for 40 µs of aggregated time. The simulations were combined with spectroscopic data, which showed that the peptide is twofold more active in promoting lipid mixing of model membranes at pH 5 than at pH 7.4. The realistic treatment of protonation introduced by the constant-pH molecular dynamics simulations revealed that low pH stabilizes a vertical membrane-spanning conformation and leads to more frequent contacts between the fusion peptide and the lipid headgroups, which may explain the increase in activity. The study also revealed that the N-terminal region is determinant for the peptide's effect on the membrane.

Studying O2 pathways in [NiFe]- and [NiFeSe]-hydrogenases.

Hydrogenases are efficient biocatalysts for H2 production and oxidation with various potential biotechnological applications.[NiFe]-class hydrogenases are highly active in both production and oxidation processes-albeit primarily biased to the latter-but suffer from being sensitive to O2.[NiFeSe] hydrogenases are a subclass of [NiFe] hydrogenases with, usually, an increased insensitivity to aerobic environments. In this study we aim to understand the structural causes of the low sensitivity of a [NiFeSe]-hydrogenase, when compared with a [NiFe] class enzyme, by studying the diffusion of O2. To unravel the differences between the two enzymes, we used computational methods comprising Molecular Dynamics simulations with explicit O2 and Implicit Ligand Sampling methodologies. With the latter, we were able to map the free energy landscapes for O2 permeation in both enzymes. We derived pathways from these energy landscapes and selected the kinetically more relevant ones with reactive flux analysis using transition path theory. These studies evidence the existence of quite different pathways in both enzymes and predict a lower permeation efficiency for O2 in the case of the [NiFeSe]-hydrogenase when compared with the [NiFe] enzyme. These differences can explain the experimentally observed lower inhibition by O2 on [NiFeSe]-hydrogenases, when compared with [NiFe]-hydrogenases. A comprehensive map of the residues lining the most important O2 pathways in both enzymes is also presented.

Synthesis and biological effects of small molecule enhancers for improved recombinant protein production in plant cell cultures.

Histone deacetylases are involved in chromatin remodelling and thus play a vital role in the epigenetic regulation of gene expression. HDAC inhibitors alter the acetylation status of histone and non-histone proteins to regulate various cellular events such as transcription. Novel HDAC inhibitors were designed and synthesised to promote higher levels of recombinant protein production in tobacco cell cultures. The effect of these chemical enhancers on the epigenetic profiles in plant cells has been evaluated by molecular docking, in vitro and in vivo studies. The addition of these novel enhancers led to an increase in histone H3 acetylation levels that promoted an increase in the accumulation levels of the recombinant protein in cell culture. These results can pave the way for the application of these enhancers to improve the production of high value products in plant cell based systems.

A LysM Domain Intervenes in Sequential Protein-Protein and Protein-Peptidoglycan Interactions Important for Spore Coat Assembly in Bacillus subtilis.

At a late stage in spore development in Bacillus subtilis, the mother cell directs synthesis of a layer of peptidoglycan known as the cortex between the two forespore membranes, as well as the assembly of a protective protein coat at the surface of the forespore outer membrane. SafA, the key determinant of inner coat assembly, is first recruited to the surface of the developing spore and then encases the spore under the control of the morphogenetic protein SpoVID. SafA has a LysM peptidoglycan-binding domain, SafALysM, and localizes to the cortex-coat interface in mature spores. SafALysM is followed by a region, A, required for an interaction with SpoVID and encasement. We now show that residues D10 and N30 in SafALysM, while involved in the interaction with peptidoglycan, are also required for the interaction with SpoVID and encasement. We further show that single alanine substitutions on residues S11, L12, and I39 of SafALysM that strongly impair binding to purified cortex peptidoglycan affect a later stage in the localization of SafA that is also dependent on the activity of SpoVE, a transglycosylase required for cortex formation. The assembly of SafA thus involves sequential protein-protein and protein-peptidoglycan interactions, mediated by the LysM domain, which are required first for encasement then for the final localization of the protein in mature spores.IMPORTANCEBacillus subtilis spores are encased in a multiprotein coat that surrounds an underlying peptidoglycan layer, the cortex. How the connection between the two layers is enforced is not well established. Here, we elucidate the role of the peptidoglycan-binding LysM domain, present in two proteins, SafA and SpoVID, that govern the localization of additional proteins to the coat. We found that SafALysM is a protein-protein interaction module during the early stages of coat assembly and a cortex-binding module at late stages in morphogenesis, with the cortex-binding function promoting a tight connection between the cortex and the coat. In contrast, SpoVIDLysM functions only as a protein-protein interaction domain that targets SpoVID to the spore surface at the onset of coat assembly.

An electrogenic redox loop in sulfate reduction reveals a likely widespread mechanism of energy conservation.

The bioenergetics of anaerobic metabolism frequently relies on redox loops performed by membrane complexes with substrate- and quinone-binding sites on opposite sides of the membrane. However, in sulfate respiration (a key process in the biogeochemical sulfur cycle), the substrate- and quinone-binding sites of the QrcABCD complex are periplasmic, and their role in energy conservation has not been elucidated. Here we show that the QrcABCD complex of Desulfovibrio vulgaris is electrogenic, as protons and electrons required for quinone reduction are extracted from opposite sides of the membrane, with a H+/e- ratio of 1. Although the complex does not act as a H+-pump, QrcD may include a conserved proton channel leading from the N-side to the P-side menaquinone pocket. Our work provides evidence of how energy is conserved during dissimilatory sulfate reduction, and suggests mechanisms behind the functions of related bacterial respiratory complexes in other bioenergetic contexts.

Study of the interactions of bovine serum albumin with a molybdenum(II) carbonyl complex by spectroscopic and molecular simulation methods.

Therapy with inhaled carbon monoxide (CO) is being tested in human clinical trials, yet the alternative use of prodrugs, CO-Releasing Molecules (CORMs), is conceptually advantageous. These molecules are designed to release carbon monoxide in specific tissues, in response to some locally expressed stimulus, where CO can trigger a cytoprotective response. The design of such prodrugs, mostly metal carbonyl complexes, must consider their ADMET profiles, including their interaction with transport plasma proteins. However, the molecular details of this interaction remain elusive. To shed light into this matter, we focused on the CORM prototype [Mo(η5-Cp)(CH2COOH)(CO)3] (ALF414) and performed a detailed molecular characterization of its interaction with bovine serum albumin (BSA), using spectroscopic and computational methods. The experimental results show that ALF414 partially quenches the intrinsic fluorescence of BSA without changing its secondary structure. The interaction between BSA and ALF414 follows a dynamic quenching mechanism, indicating that no stable complex is formed between the protein Trp residues and ALF414. The molecular dynamics simulations are in good agreement with the experimental results and confirm the dynamic and unspecific character of the interaction between ALF414 and BSA. The simulations also provide important insights into the nature of the interactions of this CORM prototype with BSA, which are dominated by hydrophobic contacts, with a contribution from hydrogen bonding. This kind of information is useful for future CORM design.

AOX1-Subfamily Gene Members in Olea europaea cv. "Galega Vulgar"-Gene Characterization and Expression of Transcripts during IBA-Induced in Vitro Adventitious Rooting.

Propagation of some Olea europaea L. cultivars is strongly limited due to recalcitrant behavior in adventitious root formation by semi-hardwood cuttings. One example is the cultivar "Galega vulgar". The formation of adventitious roots is considered a morphological response to stress. Alternative oxidase (AOX) is the terminal oxidase of the alternative pathway of the plant mitochondrial electron transport chain. This enzyme is well known to be induced in response to several biotic and abiotic stress situations. This work aimed to characterize the alternative oxidase 1 (AOX1)-subfamily in olive and to analyze the expression of transcripts during the indole-3-butyric acid (IBA)-induced in vitro adventitious rooting (AR) process. OeAOX1a (acc. no. MF410318) and OeAOX1d (acc. no. MF410319) were identified, as well as different transcript variants for both genes which resulted from alternative polyadenylation events. A correlation between transcript accumulation of both OeAOX1a and OeAOX1d transcripts and the three distinct phases (induction, initiation, and expression) of the AR process in olive was observed. Olive AOX1 genes seem to be associated with the induction and development of adventitious roots in IBA-treated explants. A better understanding of the molecular mechanisms underlying the stimulus needed for the induction of adventitious roots may help to develop more targeted and effective rooting induction protocols in order to improve the rooting ability of difficult-to-root cultivars.