Use of the KlADH3 promoter for the quantitative production of the murine PDE5A isoforms in the yeast Kluyveromyces lactis

Background: Phosphodiesterases (PDE) are a superfamily of enzymes that hydrolyse cyclic nucleotides (cAMP/ cGMP), signal molecules in transduction pathways regulating crucial aspects of cell life. PDEs regulate the intensity and duration of the cyclic nucleotides signal modulating the downstream biological efect. Due to this critical role associated with the extensive distribution and multiplicity of isozymes, the 11 mammalian families (PDE1 to PDE11) constitute key therapeutic targets. PDE5, one of these cGMP-specifc hydrolysing families, is the molecular target of several well known drugs used to treat erectile dysfunction and pulmonary hypertension. Kluyveromyces lactis, one of the few yeasts capable of utilizing lactose, is an attractive host alternative to Saccharomyces cerevisiae for heterologous protein production. Here we established K. lactis as a powerful host for the quantitative production of the murine PDE5 isoforms. Results: Using the promoter of the highly expressed KlADH3 gene, multicopy plasmids were engineered to produce the native and recombinant Mus musculus PDE5 in K. lactis. Yeast cells produced large amounts of the purifed A1, A2 and A3 isoforms displaying Km, Vmax and Sildenafl inhibition values similar to those of the native murine enzymes. PDE5 whose yield was nearly 1 mg/g wet weight biomass for all three isozymes (30 mg/L culture), is well tolerated by K. lactis cells without major growth defciencies and interferences with the endogenous cAMP/cGMP signal transduction pathways. Conclusions: To our knowledge, this is the frst time that the entire PDE5 isozymes family containing both regulatory and catalytic domains has been produced at high levels in a heterologous eukaryotic organism. K. lactis has been shown to be a very promising host platform for large scale production of mammalian PDEs for biochemical and structural studies and for the development of new specifc PDE inhibitors for therapeutic applications in many pathologies

Use of the KlADH4 promoter for ethanol-dependent production of recombinant human serum albumine in Kluyveromyces lactis

KlADH4 is a gene of Kluyveromyces lactis encoding a mitochondrial alcohol dehydrogenase activity which is specifically induced by ethanol. The promoter of this gene was used for the expression of heterologous proteins in K. lactis, a very promising organism which can be used as an alternative host to Saccharomyces cerevisiae due to its good secretory properties. In this paper we report the ethanol-driven expression in K. lactis of the bacterial beta-glucuronidase and of the human serum albumin (HSA) genes under the control of the KlADH4 promoter. In particular, we studied the extracellular production of recombinant HSA (rHSA) with integrative and replicative vectors and obtained a significant increase in the amount of the protein with multicopy vectors, showing that no limitation of KlADH4 trans-acting factors occurred in the cells. By deletion analysis of the promoter, we identified an element (UASE) which is sufficient for the induction of KlADH4 by ethanol and, when inserted in the respective promoters, allows ethanol-dependent activation of other yeast genes, such as PGK and LAC4. We also analyzed the effect of medium composition on cell growth and protein secretion. A clear improvement in the production of the recombinant protein was achieved by shifting from batch cultures (0.3 g/liter) to fed-batch cultures (1 g/liter) with ethanol as the preferred carbon source

Characterization of the transcription factor encoding gene, KlADR1: metabolic role in Kluyveromyces lactis and expression in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, Adr1 is a zinc-finger transcription factor involved in the transcriptional activation of ADH2. Deletion of KlADR1, its putative ortholog in Kluyveromyces lactis, led to reduced growth in glycerol, oleate and yeast extract-peptone medium suggesting, as in S. cerevisiae, its requirement for glycerol, fatty acid and nitrogen utilization. Moreover, growth comparison on yeast extract and peptone plates showed in K. lactis a KlAdr1-dependent growth trait not present in S. cerevisiae, indicating different metabolic roles of the two factors in their environmental niches. KlADR1 is required for growth under respiratory and fermentative conditions like KlADH, alcohol dehydrogenase genes necessary for metabolic adaptation during the growth transition. Using in-gel native alcohol dehydrogenase assay, we showed that this factor affected the Adh pattern by altering the balance between these activities. Since the activity most affected by KlAdr1 is KlAdh3, a deletion analysis of the KlADH3 promoter allowed the isolation of a DNA fragment through which KlAdr1 modulated its expression. The expression of the KlADR1-GFP gene allowed the intracellular localization of the factor in K. lactis and S. cerevisiae, suggesting in the two yeasts a common mechanism of KlAdr1 translocation under fermentative and respiratory conditions. Finally, the chimeric Kl/ScADR1 gene encoding the zinc- finger domains of KlAdr1 fused to the transactivating domains of the S. cerevisiae factor activated in Scadr1D the transcription of ADH2 in a ScAdr1-dependent fashion

Role of yUbp8 in Mitochondria and Hypoxia Entangles the Finding of Human Ortholog Usp22 in the Glioblastoma Pseudo-Palisade Microlayer

KAT Gcn5 and DUB Ubp8 are required for respiration and mitochondria functions in budding yeast, and in this study we show that loss of respiratory activity is acquired over time. Interestingly, we show that absence of Ubp8 allows cells to grow in hypoxic conditions with altered mitophagy. Comparatively, the aggressive glioblastoma (GBM) multiforme tumor shows survival mechanisms able to overcome hypoxia in the brain. Starting from yeast and our findings on the role of Ubp8 in hypoxia, we extended our analysis to the human ortholog and signature cancer gene Usp22 in glioblastoma tumor specimens. Here we demonstrate that Usp22 is localized and overexpressed in the pseudo-palisade tissue around the necrotic area of the tumor. In addition, Usp22 colocalizes with the mitophagy marker Parkin, indicating a link with mitochondria function in GBM. Collectively, this evidence suggests that altered expression of Usp22 might provide a way for tumor cells to survive in hypoxic conditions, allowing the escape of cells from the necrotic area toward vascularized tissues. Collectively, our experimental data suggest a model for a possible mechanism of uncontrolled proliferation and invasion in glioblastoma

Arabidopsis thaliana sirtuins control proliferation and glutamate dehydrogenase activity

Sirtuins are part of a gene family of NAD-dependent deacylases that act on histone and non-histone proteins and control a variety of activities in all living organisms. Their roles are mainly related to energy metabolism and include lifetime regulation, DNA repair, stress resistance, and proliferation. A large amount of knowledge concerning animal sirtuins is available, but data about their plant counterparts are scarce. Plants possess few sirtuins that have, like in animals, a recognized role in stress defense and metabolism regulation. However, engagement in proliferation control, which has been demonstrated for mammalian sirtuins, has not been reported for plant sirtuins so far. In this work, srt1 and srt2 Arabidopsis mutant seedlings have been used to evaluate in vivo the role of sirtuins in cell proliferation and regulation of glutamate dehydrogenase, an enzyme demonstrated to be involved in the control of cell cycle in SIRT4-defective human cells. Moreover, bioinformatic analyses have been performed to elucidate sequence, structure, and function relationships between Arabidopsis sirtuins and between each of them and the closest mammalian homolog. We found that cell proliferation and GDH activity are higher in mutant seedlings, suggesting that both sirtuins exert a physiological inhibitory role in these processes. In addition, mutant seedlings show plant growth and root system improvement, in line with metabolic data. Our data also indicate that utilization of an easy to manipulate organism, such as Arabidopsis plant, can help to shed light on the molecular mechanisms underlying the function of genes present in interkingdom species

Relationship between low Ankle-Brachial Index and rapid renal function decline in patients with atrial fibrillation: A prospective multicentre cohort study

OBJECTIVE: To investigate the relationship between Ankle-Brachial Index (ABI) and renal function progression in patients with atrial fibrillation (AF). DESIGN: Observational prospective multicentre cohort study. SETTING:Atherothrombosis Center of I Clinica Medica of 'Sapienza' University of Rome; Department of Medical and Surgical Sciences of University Magna Græcia of Catanzaro; Atrial Fibrillation Registry for Ankle-Brachial Index Prevalence Assessment-Collaborative Italian Study. PARTICIPANTS: 897 AF patients on treatment with vitamin K antagonists. MAIN OUTCOME MEASURES: The relationship between basal ABI and renal function progression, assessed by the estimated Glomerular Filtration Rate (eGFR) calculated with the CKD-EPI formula at baseline and after 2 years of follow-up. The rapid decline in eGFR, defined as a decline in eGFR >5 mL/min/1.73 m(2)/year, and incident eGFR<60 mL/min/1.73 m(2) were primary and secondary end points, respectively. RESULTS: Mean age was 71.8±9.0 years and 41.8% were women. Low ABI (ie, ≤0.90) was present in 194 (21.6%) patients. Baseline median eGFR was 72.7 mL/min/1.73 m(2), and 28.7% patients had an eGFR60 mL/min/1.73 m(2), 153 (23.9%) had a reduction of the eGFR <60 mL/min/1.73 m(2). ABI ≤0.90 was also an independent predictor for incident eGFR<60 mL/min/1.73 m(2) (HR 1.851, 95% CI 1.205 to 2.845, p=0.005). CONCLUSIONS: In patients with AF, an ABI ≤0.90 is independently associated with a rapid decline in renal function and incident eGFR<60 mL/min/1.73 m(2). ABI measurement may help identify patients with AF at risk of renal function deterioration

Frequency of left ventricular hypertrophy in non-valvular atrial fibrillation

Left ventricular hypertrophy (LVH) is significantly related to adverse clinical outcomes in patients at high risk of cardiovascular events. In patients with atrial fibrillation (AF), data on LVH, that is, prevalence and determinants, are inconsistent mainly because of different definitions and heterogeneity of study populations. We determined echocardiographic-based LVH prevalence and clinical factors independently associated with its development in a prospective cohort of patients with non-valvular (NV) AF. From the "Atrial Fibrillation Registry for Ankle-brachial Index Prevalence Assessment: Collaborative Italian Study" (ARAPACIS) population, 1,184 patients with NVAF (mean age 72 \ub1 11 years; 56% men) with complete data to define LVH were selected. ARAPACIS is a multicenter, observational, prospective, longitudinal on-going study designed to estimate prevalence of peripheral artery disease in patients with NVAF. We found a high prevalence of LVH (52%) in patients with NVAF. Compared to those without LVH, patients with AF with LVH were older and had a higher prevalence of hypertension, diabetes, and previous myocardial infarction (MI). A higher prevalence of ankle-brachial index 640.90 was seen in patients with LVH (22 vs 17%, p = 0.0392). Patients with LVH were at significantly higher thromboembolic risk, with CHA2DS2-VASc 652 seen in 93% of LVH and in 73% of patients without LVH (p &lt;0.05). Women with LVH had a higher prevalence of concentric hypertrophy than men (46% vs 29%, p = 0.0003). Logistic regression analysis demonstrated that female gender (odds ratio [OR] 2.80, p &lt;0.0001), age (OR 1.03 per year, p &lt;0.001), hypertension (OR 2.30, p &lt;0.001), diabetes (OR 1.62, p = 0.004), and previous MI (OR 1.96, p = 0.001) were independently associated with LVH. In conclusion, patients with NVAF have a high prevalence of LVH, which is related to female gender, older age, hypertension, and previous MI. These patients are at high thromboembolic risk and deserve a holistic approach to cardiovascular prevention

Ethanol-induced and glucose-insensitive alcohol dehydrogenase activity in the yeast Kluyveromyces lactis.

The alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis is encoded by four ADH genes. In this paper we report evidence that at least three of these genes are transcribed and translated into protein. KIADH1 and KIADH2, which encode cytoplasmic activities, are preferentially expressed in glucose-grown cells with respect to ethanol-grown cells. KIADH4, which encodes one of the two activities localized within mitochondria, is induced at the transcriptional level in the presence of ethanol as is the ADH2 gene in Saccharomyces cerevisiae. However the regulation of the expression of the K. lactis gene is completely different from that of ADH2 and of other known ADH genes in that KIADH4 is insensitive to glucose repression and is not expressed on non-fermentable carbon sources other than ethanol. This kind of regulation can be clearly observed in non-fermenting strains, where the induction of KIADH4 is dependent on the addition of ethanol to the medium. On the contrary, in fermenting strains KIADH4 is always induced by ethanol or acetaldehyde produced endocellularly and this results in constitutive expression of the gene also in the presence of glucose. The mitochondrial localization of the activity encoded by KIADH4 and the peculiar regulation of this gene could be related to the fact that K. lactis is a petite negative yeast in which some mitochondrial functions seem to be essential for cell viability

Alcohol dehydrogenase (ADH) isozymes in Kluyveromyces lactis: detection by activity.

Chapter 3