Cardosin A endocytosis mediated by integrin leads to lysosome leakage and apoptosis of epithelial cells.

Cardosin A is an aspartic protease present in large amount in the pistils of cardoon flowers. This protease is known to contain an -Arg-Gly-Asp- (RGD) motif located on the molecular surface. In this study, we found that isolated recombinant cardosin A attached to human epithelial cells A549, mediated by the binding of its RGD motif to cell surface integrins. The cell bound cardosin A was internalized to endosomes and lysosomes and triggered the permeability of lysosomal membrane leading to apoptosis of the epithelial cells. These events are identical to those observed for three RGD-containing aspartic proteases, Saps 4-6, secreted by Candida albicans. Such a process, which has been called the Trojan Horse mechanism, is believed to benefit the invasion of C. albican into the epithelium of the host. The location of the RGD motifs of cardosin A and Saps 4-6 are on the opposite ends of the homologous three-dimensional structures, suggesting that the Trojan Horse mechanism is insensitive to the RGD position. Current finding also suggests that cardosin A may have a defensive function against the ingestion of cardoon flowers by human, insects, and other herbivores.

An atypical aspartic protease modulates lateral root development in Arabidopsis thaliana.

Few atypical aspartic proteases (APs) present in plants have been functionally studied to date despite having been implicated in developmental processes and stress responses. Here we characterize a novel atypical AP that we name Atypical Aspartic Protease in Roots 1 (ASPR1), denoting its expression in Arabidopsis roots. Recombinant ASPR1 produced by transient expression in Nicotiana benthamiana was active and displayed atypical properties, combining optimum acidic pH, partial sensitivity to pepstatin, pronounced sensitivity to redox agents, and unique specificity preferences resembling those of fungal APs. ASPR1 overexpression suppressed primary root growth and lateral root development, implying a previously unknown biological role for an AP. Quantitative comparison of wild-type and aspr1 root proteomes revealed deregulation of proteins associated with both reactive oxygen species and auxin homeostasis in the mutant. Together, our findings on ASPR1 reinforce the diverse pattern of enzymatic properties and biological roles of atypical APs and raise exciting questions on how these distinctive features impact functional specialization among these proteases.

Functional and structural characterization of synthetic cardosin B-derived rennet.

The potential of using a synthetic cardosin-based rennet in cheese manufacturing was recently demonstrated with the development and optimization of production of a recombinant form of cardosin B in Kluyveromyces lactis. With the goal of providing a more detailed characterization of this rennet, we herein evaluate the impact of the plant-specific insert (PSI) on cardosin B secretion in this yeast, and provide a thorough analysis of the specificity requirements as well as the biochemical and structural properties of the isolated recombinant protease. We demonstrate that the PSI domain can be substituted by different linker sequences without substantially affecting protein secretion and milk clotting activity. However, the presence of small portions of the PSI results in dramatic reductions of secretion yields in this heterologous system. Kinetic characterization and specificity profiling results clearly suggest that synthetic cardosin B displays lower catalytic efficiency and is more sequence selective than native cardosin B. Elucidation of the structure of synthetic cardosin B confirms the canonical fold of an aspartic protease with the presence of two high mannose-type, N-linked glycan structures; however, there are some differences in the conformation of the flap region when compared to cardosin A. These subtle variations in catalytic properties and the more stringent substrate specificity of synthetic cardosin B help to explain the observed suitability of this rennet for cheese production.

Biophysical characterization of laforin-carbohydrate interaction.

Laforin is a human dual-specificity phosphatase (DSP) involved in glycogen metabolism regulation containing a carbohydrate-binding module (CBM). Mutations in the gene coding for laforin are responsible for the development of Lafora disease, a progressive fatal myoclonus epilepsy with early onset, characterized by the intracellular deposition of abnormally branched, hyperphosphorylated insoluble glycogen-like polymers, called Lafora bodies. Despite the known importance of the CBM domain of laforin in the regulation of glycogen metabolism, the molecular mechanism of laforin-glycogen interaction is still poorly understood. Recently, the structure of laforin with bound maltohexaose was determined and despite the importance of such breakthrough, some molecular interaction details remained missing. We herein report a thorough biophysical characterization of laforin-carbohydrate interaction using soluble glycans. We demonstrated an increased preference of laforin for the interaction with glycans with higher order of polymerization and confirmed the importance of tryptophan residues for glycan interaction. Moreover, and in line with what has been described for other CBMs and lectins, our results confirmed that laforin-glycan interactions occur with a favourable enthalpic contribution counter-balanced by an unfavourable entropic contribution. The analysis of laforin-glycan interaction through the glycan side by saturation transfer difference (STD)-NMR has shown that the CBM-binding site can accommodate between 5 and 6 sugar units, which is in line with the recently obtained crystal structure of laforin. Overall, the work in the present study complements the structural characterization of laforin and sheds light on the molecular mechanism of laforin-glycan interaction, which is a pivotal requisite to understand the physiological and pathological roles of laforin.

RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel bona fide member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.

Structure of RC1339/APRc from Rickettsia conorii, a retropepsin-like aspartic protease.

The crystal structures of two constructs of RC1339/APRc from Rickettsia conorii, consisting of either residues 105-231 or 110-231 followed by a His tag, have been determined in three different crystal forms. As predicted, the fold of a monomer of APRc resembles one-half of the mandatory homodimer of retroviral pepsin-like aspartic proteases (retropepsins), but the quaternary structure of the dimer of APRc differs from that of the canonical retropepsins. The observed dimer is most likely an artifact of the expression and/or crystallization conditions since it cannot support the previously reported enzymatic activity of this bacterial aspartic protease. However, the fold of the core of each monomer is very closely related to the fold of retropepsins from a variety of retroviruses and to a single domain of pepsin-like eukaryotic enzymes, and may represent a putative common ancestor of monomeric and dimeric aspartic proteases.

Engineering a cardosin B-derived rennet for sheep and goat cheese manufacture.

Different sheep and goat cheeses with world-renowned excellence are produced using aqueous extracts of Cynara cardunculus flowers as coagulants. However, the use of this vegetable rennet is mostly limited to artisanal scale production, and no effective solutions to large-scale industrial applications have been reported so far. In this sense, the development of a synthetic rennet based on the most abundant cardoon milk-clotting enzymes (cardosins) would emerge as a solution for scalability of production and for application of these proteases as alternative rennets in dairy industry. In this work, we report the development of a new cardosin B-derived rennet produced in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. Using a stepwise optimization strategy-consisting of culture media screening, complemented with a protein engineering approach with removal of the plant-specific domain, and a codon optimization step-we successfully improved cardosin B production yield (35×) in K. lactis. We demonstrated that the secreted enzyme displays similar proteolytic properties, such as casein digestion profiles as well as optimum pH (pH 4.5) and temperature (40 °C), with those of native cardosin B. From this optimization process resulted the rennet preparation Vegetable Rennet (VRen), requiring no downstream protein purification steps. The effectiveness of VRen in cheese production was demonstrated by manufacturing sheep, goat, and cow cheeses. Interestingly, the use of VRen resulted in a higher cheese yield for all three types of cheese when compared with synthetic chymosin. Altogether, these results clearly position VRen as an alternative/innovative coagulant for the cheese-making industry.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing.

BACKGROUND: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. RESULTS: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. CONCLUSIONS: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Establishing the yeast Kluyveromyces lactis as an expression host for production of the saposin-like domain of the aspartic protease cirsin.

Typical plant aspartic protease zymogens comprise a characteristic and plant-specific insert (PSI). PSI domains can interact with membranes, and a role as a defensive weapon against pathogens has been proposed. However, the potential of PSIs as antimicrobial agents has not been fully investigated and explored yet due to problems in producing sufficient amounts of these domains in bacteria. Here, we report the development of an expression platform for the production of the PSI domain of cirsin in the generally regarded as safe (GRAS) yeast Kluyveromyces lactis. We successfully generated K. lactis transformants expressing and secreting significant amounts of correctly processed and glycosylated PSI, as well as its nonglycosylated mutant. A purification protocol with protein yields of ∼4.0 mg/liter was established for both wild-type and nonglycosylated PSIs, which represents the highest reported yield for a nontagged PSI domain. Subsequent bioactivity assays targeting phytopathogenic fungi indicated that the PSI of cirsin is produced in a biologically active form in K. lactis and provided clear evidence for its antifungal activity. This yeast expression system thereby emerges as a promising production platform for further exploring the biotechnological potential of these plant saposin-like proteins.

Dose-dependent inhibition of BACE-1 by the monoterpenoid 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone in cellular and mouse models of Alzheimer's disease.

BACE-1 is an aspartic protease involved in the conversion of amyloid precursor protein (APP) to amyloid-ß (Aß) in vivo, which is one of the key steps in the development and progression of Alzheimer's disease. In a previous screening procedure for inhibitors of BACE-1 activity, the oil of Lavandula luisieri was identified as the most potent among several essential oils. The inhibitory effect of this essential oil on Aß production was also demonstrated in a cellular assay. The composition of the volatile oil and the isolation of the compound responsible for the inhibitory activity were also reported. The present work focused on the characterization of the inhibition of BACE-1 by this active compound, a monoterpene necrodane ketone, 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1), with assessment of its Ki value and the type of inhibition. The dose-related effects of the compound were also evaluated using two different cell lines, with determinations of the respective EC50 values. The entire oil and the 2,3,4,4-tetramethyl-5-methylenecyclopent-2-enone (1) were tested on a triple transgenic mouse model of Alzheimer's disease. The overall results showed that compound 1 displayed a dose-dependent inhibition of BACE-1 in cellular and mouse models of Alzheimer's disease and is therefore capable of passing through cellular membranes and the blood-brain barrier.

Genetic variants in ICAM1, PPARGC1A and MTHFR are potentially associated with different phenotypes of diabetic retinopathy.

PURPOSE: To explore phenotype-genotype correlations that may contribute to a better understanding of diabetic retinopathy (DR). PROCEDURES: An exploratory association study was performed to identify genetic variants associated with non-proliferative DR (NPDR) in 307 type 2 diabetic patients who were previously stratified into 3 different phenotypes of NPDR progression. The 307 patients were genotyped for 174 single nucleotide polymorphisms of 11 candidate genes (ACE, AGER, AKR1B1, ICAM1, MTHFR, NOS1, NOS3, PPARGC1A, TGFB1, TNF and VEGFA). RESULTS: Significant associations were observed for PPARGC1A rs16874120 with phenotype A (odds ratio, OR = 0.60, 95% confidence interval, CI 0.36-0.99), ICAM1 rs1801714 with phenotype B (OR = 3.32, 95% CI 1.05-10.50) and both PPARGC1A rs10213440 (OR = 2.00, 95% CI 1.07-3.73) and MTHFR rs1801133 (OR = 1.84, 95% CI 1.08-3.11) with phenotype C. CONCLUSIONS: RESULTS indicate that specific gene variants in ICAM1, PPARGC1A and MTHFR are associated with different NPDR phenotypes, being likely candidates to explain different disease mechanisms underlying the different phenotypes. This is the first study to show correlations between specific gene variants and NPDR phenotypes, opening new perspectives on DR.

Chlapsin, a chloroplastidial aspartic proteinase from the green algae Chlamydomonas reinhardtii.

Aspartic proteinases have been extensively characterized in land plants but up to now no evidences for their presence in green algae group have yet been reported in literature. Here we report on the identification of the first (and only) typical aspartic proteinase from Chlamydomonas reinhardtii. This enzyme, named chlapsin, was shown to maintain the primary structure organization of typical plant aspartic proteinases but comprising distinct features, such as similar catalytic motifs DTG/DTG resembling those from animal and microbial counterparts, and an unprecedentedly longer plant specific insert domain with an extra segment of 80 amino acids, rich in alanine residues. Our results also demonstrated that chlapsin accumulates in Chlamydomonas chloroplast bringing this new enzyme to a level of uniqueness among typical plant aspartic proteinases. Chlapsin was successfully expressed in Escherichia coli and it displayed the characteristic enzymatic properties of typical aspartic proteinases, like optimum activity at acidic pH and complete inhibition by pepstatin A. Another difference to plant aspartic proteinases emerged as chlapsin was produced in an active form without its putative prosegment domain. Moreover, recombinant chlapsin showed a restricted enzymatic specificity and a proteolytic activity influenced by the presence of redox agents and nucleotides, further differentiating it from typical plant aspartic proteinases and anticipating a more specialized/regulated function for this Chlamydomonas enzyme. Taken together, our results revealed a pattern of complexity for typical plant aspartic proteinases in what concerns sequence features, localization and biochemical properties, raising new questions on the evolution and function of this vast group of plant enzymes.

Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10(4) s(-1) m(-1) against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.

Self-assembled dextrin nanogel as protein carrier: controlled release and biological activity of IL-10.

Interleukin-10 (IL-10) is an anti-inflammatory cytokine, which active form is a non-covalent homodimer. Given the potential of IL-10 for application in various medical conditions, it is essential to develop systems for its effective delivery. In previous work, it has been shown that a dextrin nanogel effectively incorporated and stabilized rIL-10, enabling its release over time. In this work, the delivery system based on dextrin nanogels was further analyzed. The biocompatibility of the nanogel was comprehensively analyzed, through cytotoxicity (lactate dehydrogenase (LDH) release, MTS, Live, and Dead) and genotoxicity (comet) assays. The release profile of rIL-10 and its biological activity were evaluated in vivo, using C57BL/6 mice. Although able to maintain a stable concentration of IL-10 for at least 4 h in mice serum, the amount of protein released was rather low. Despite this, the amount of rIL-10 released from the complex was biologically active inhibiting TNF-α production, in vivo, by LPS-challenged mice. In spite of the significant stabilization achieved using the nanogel, rIL-10 still denatures rather quickly. An additional effort is thus necessary to develop an effective delivery system for this cytokine, able to release active protein over longer periods of time. Nevertheless, the good biocompatibility, the protein stabilization effect and the ability to perform as a carrier with controlled release suggest that self-assembled dextrin nanogels may be useful protein delivery systems.

Metabolic pathways in sporadic colorectal carcinogenesis: A new proposal.

Given the reports made about geographical differences in Colorectal Cancer (CRC) occurrence, suggesting a link between dietary habits, genes and cancer risk, we hypothesise that there are four fundamental metabolic pathways involved in diet-genes interactions, directly implicated in colorectal carcinogenesis: folate metabolism; lipid metabolism; oxidative stress response; and inflammatory response. Supporting this hypothesis are the evidence given by the significant associations between several diet-genes polymorphisms and CRC, namely: MTHFR, MTR, MTRR and TS (involved in folate metabolism); NPY, APOA1, APOB, APOC3, APOE, CETP, LPL and PON1 (involved in lipid metabolism); MNSOD, SOD3, CAT, GSTP1, GSTT1 and GSTM1 (involved in oxidative stress response); and IL-1, IL-6, TNF-α, and TGF-ß (involved in inflammatory response). We also highlight the association between some foods/nutrients/nutraceuticals that are important in CRC prevention or treatment and the four metabolic pathways proposed, and the recent results of genome-wide association studies, both assisting our hypothesis. Finally, we propose a new line of investigation with larger studies, using accurate dietary biomarkers and investigating the four metabolic pathways genes simultaneously. This line of investigation will be essential to understand the full complexity of the association between nature and nurture in CRC and perhaps in other types of cancers. Only with this in-depth knowledge will it be possible to make personalised nutrition recommendations for disease prevention and management.

Escherichia coli expression, refolding and characterization of human laforin.

Laforin is a unique human dual-specificity phosphatase as it contains an amino terminal carbohydrate binding module (CBM). Laforin gene mutations lead to Lafora disease, a progressive myoclonus epilepsy with an early fatal issue. Previous attempts to produce recombinant laforin faced various difficulties, namely the appearance of protein inclusion bodies, the contamination with bacterial proteins and a high tendency of the protein to aggregate, despite the use of fusion tags to improve solubility and ease the purification process. In this work, we have expressed human laforin in Escherichia coli in the form of inclusion bodies devoid of any fusion tags. After a rapid dilution refolding step, the protein was purified by two chromatographic steps, yielding 5-7mg of purified protein per liter of bacterial culture. The purified protein was shown to have the kinetic characteristics of a dual-specificity phosphatase, and a functional carbohydrate binding module. With this protocol, we were able for the first time, to produce and purify laforin without fusion tags in the amounts traditionally needed for the crystallographic structural studies paving the way to the understanding of the molecular mechanisms of laforin activity and to the development of novel therapies for Lafora disease.

Expression of the functional carbohydrate-binding module (CBM) of human laforin.

Laforin is a human protein associated with the glycogen metabolism, composed of two structurally and functionally independent domains: a phosphatase catalytic domain and a substrate-binding module with glycogen and starch affinity. The main goal of this work is the development of a methodology for the expression of the so far poorly characterized carbohydrate-binding module (CBM) of laforin, allowing its study and development of biomedical applications. The laforin's CBM sequence was originally cloned by PCR from a human muscle cDNA library. The recombinant protein, containing laforin's CBM fused to an Arg-Gly-Asp sequence (RGD), was cloned and expressed using vector pET29a and recovered as inclusion bodies (IBs). Refolding of the IBs allowed the purification of soluble, dimeric and functional protein, according to adsorption assays using starch and glycogen. Several other experimental approaches, using both bacteria and yeast, were unsuccessfully tested, pointing towards the difficulties in producing the heterologous protein. Indeed, this is the first work reporting the production of the functional CBM from human laforin.

African swine fever virus p10 protein exhibits nuclear import capacity and accumulates in the nucleus during viral infection.

African swine fever virus (ASFV), a large enveloped DNA-containing virus, infects domestic and wild pigs, and multiplies in soft ticks, causing an economically relevant hemorrhagic disease. Evaluation of the nuclear import ability of ASFV p10 protein was the major purpose of the present work. Two approaches were used to determine if p10 protein is imported into the nucleus by an active process: a yeast-based nuclear import assay and the determination of the subcellular localization of p10 protein in mammalian cells by fluorescence microscopy. The results obtained clearly demonstrate that p10 protein is actively imported into the nucleus, both in yeast and mammalian cells. Experiments aiming at identifying the critical residues responsible for the nuclear import of ASFV p10 protein indicate that the amino acids comprised between the positions 71 and 77 are important, although not sufficient, for the protein active nuclear import. In ASFV-infected cells, the p10 protein strongly accumulates in the nucleus at late times post-infection, indicating that p10 protein may accomplish an important function inside the nucleus during the late phase of the viral replication cycle.

Miniature cheeses made with blends of chymosin and a vegetable rennet from flowers of Silybum marianum: Enzymatic characterization of the flower-coagulant peptidase.

Binary blends of S. marianum-flower extract and chymosin, as coagulant preparations, enabled the manufacture of miniature cheeses with distinctive characteristics compared to those of chymosin-renneted cheeses. The physicochemical parameters, sensory attributes of the cheeses, and in-vitro water-soluble antioxidant activity were analyzed and compared to those properties obtained from control chymosin-renneted cheeses. The preponderant proteolytic constituent in the flower extract was isolated in a two-step-purification protocol. The thus purified aspartic peptidase was maximally active at acidic pHs and exhibited a preference for peptide bonds between hydrophobic residues. Enzymologic characterization revealed differences in the kinetic parameters and specificity compared to other enzymes employed, such as rennet. S. marianum-flower extract, as a source of peptidase with distinctive characteristics, is a suitable substitute for chymosin in miniature-cheese production. The addition of vegetable rennet contributed to the development of an intense aroma and conferred antioxidant activity to the cheeses and wheys.

The draft genome sequence of cork oak.

Cork oak (Quercus suber) is native to southwest Europe and northwest Africa where it plays a crucial environmental and economical role. To tackle the cork oak production and industrial challenges, advanced research is imperative but dependent on the availability of a sequenced genome. To address this, we produced the first draft version of the cork oak genome. We followed a de novo assembly strategy based on high-throughput sequence data, which generated a draft genome comprising 23,347 scaffolds and 953.3 Mb in size. A total of 79,752 genes and 83,814 transcripts were predicted, including 33,658 high-confidence genes. An InterPro signature assignment was detected for 69,218 transcripts, which represented 82.6% of the total. Validation studies demonstrated the genome assembly and annotation completeness and highlighted the usefulness of the draft genome for read mapping of high-throughput sequence data generated using different protocols. All data generated is available through the public databases where it was deposited, being therefore ready to use by the academic and industry communities working on cork oak and/or related species.