Francesca Morandini

Non‐food/feed seeds as biofactories for the high‐yield production of recombinant pharmaceuticals

We describe an attractive cloning system for the seed-specific expression of recombinant proteins using three non-food/feed crops. A vector designed for direct subcloning by Gateway® recombination was developed and tested in Arabidopsis, tobacco and petunia plants for the production of a chimeric form (GAD67/65) of the 65 kDa isoform of glutamic acid decarboxylase (GAD65). GAD65 is one of the major human autoantigens involved in type 1 diabetes (T1D). The murine anti-inflammatory cytokine interleukin-10 (IL-10) was expressed with the described system in Arabidopsis and tobacco, whereas proinsulin, another T1D major autoantigen, was expressed in Arabidopsis. The cost-effective production of these proteins in plants could allow the development of T1D prevention strategies based on the induction of immunological tolerance. The best yields were achieved in Arabidopsis seeds, where GAD67/65 reached 7.7% of total soluble protein (TSP), the highest levels ever reported for this protein in plants. IL-10 and proinsulin reached 0.70% and 0.007% of TSP, respectively, consistent with levels previously reported in other plants or tissues. This versatile cloning vector could be suitable for the high-throughput evaluation of expression levels and stability of many valuable and difficult to produce proteins.

Optimizing the purification and analysis of miRNAs from urinary exosomes

Abstract Background: Exosomes are cytoplasm containing vesicles released by many cells that can be found in several biological fluids including urine. Urinary exosomes are released from every segment of the nephron, are detectable in urine, constitutively contain RNA (small RNAs and mRNAs) and harbor unique subset of proteins, reflecting their cellular source. Methods: With the aim of establishing the optimal protocol for high throughput analysis of exosomal miRNAs, we compared three different urinary exosomes isolation methods and six RNA extraction techniques. Exosomal RNA yield, size and quality were assessed respectively by specific staining with fluorescent dye, capillary electrophoresis and analysis of spectrophotometric parameters. MiRNAs detection and abundance was determined by RT-qPCR. Results: Among the exosomes isolation methods, Ultrafiltration resulted to be the most suited. The highest exosomal RNA yield quantified by RiboGreen® staining was obtained with the combination of TRI Reagent™ with miRNeasy®, followed by TRI Reagent™, SeraMir™, miRCURY™, mirVana™ and miRNeasy®; but after a multivariate analysis, SeraMir™ scored as the method of choice in terms of miRNA yield, purity and RT-qPCR miRNAs quantification accuracy. Storage conditions were also analyzed, showing that the relative abundance of urinary exosomal miRNAs is not influenced by urine freezing. Conclusions: The selection of appropriate urinary exosomal miRNA isolation method was dependent on various validation results. Ultrafiltration in combination with SeraMir™ exoRNA columns represents the optimal procedure for a rapid, cost-effective and efficient purification of miRNAs from urinary exosomes, perfectly suited for further applicative research in the field of miRNAs in kidney physiology and pathology.

Recombinant human GAD65 accumulates to high levels in transgenic tobacco plants when expressed as an enzymatically inactive mutant

The 65-kDa isoform of glutamic acid decarboxylase (GAD65) is the major autoantigen implicated in the development of type 1 diabetes mellitus (T1DM). The bulk manufacture of GAD65 is a potential issue in the fight against T1DM but current production platforms are expensive. We show that a catalytically inactive form of GAD65 (GAD65mut) accumulates at up to 2.2% total soluble protein in transgenic tobacco leaves, which is more than 10-fold the levels achieved with active GAD65, yet the protein retains the immunogenic properties required to treat T1DM. This higher yield was found to be a result of a higher rate of protein synthesis and not transcript availability or protein stability. We found that targeting GAD65 to the endoplasmic reticulum, a strategy that increases the accumulation of many recombinant proteins expressed in plants, did not improve production of GAD65mut. The production of a catalytically inactive autoantigen that retains its immunogenic properties could be a useful strategy to provide high-quality therapeutic protein for treatment of autoimmune T1DM.

Protein body formation in the endoplasmic reticulum as an evolution of storage protein sorting to vacuoles: insights from maize γ-zein

The albumin and globulin seed storage proteins present in all plants accumulate in storage vacuoles. Prolamins, which are the major proteins in cereal seeds and are present only there, instead accumulate within the endoplasmic reticulum (ER) lumen as very large insoluble polymers termed protein bodies. Inter-chain disulfide bonds play a major role in polymerization and insolubility of many prolamins. The N-terminal domain of the maize prolamin 27 kD γ-zein is able to promote protein body formation when fused to other proteins and contains seven cysteine residues involved in inter-chain bonds. We show that progressive substitution of these amino acids with serine residues in full length γ-zein leads to similarly progressive increase in solubility and availability to traffic from the ER along the secretory pathway. Total substitution results in very efficient secretion, whereas the presence of a single cysteine is sufficient to promote partial sorting to the vacuole via a wortmannin-sensitive pathway, similar to the traffic pathway of vacuolar storage proteins. We propose that the mechanism leading to accumulation of prolamins in the ER is a further evolutionary step of the one responsible for accumulation in storage vacuoles.

Apparent Mineralocorticoid Excess by a Novel Mutation and Epigenetic Modulation by HSD11B2 Promoter Methylation

CONTEXT Apparent mineralocorticoid excess (AME) is a rare autosomal recessive disease resulting from mutations within the hydroxysteroid (11β-dehydrogenase2 [HSD11B2]) gene causing a prominent mineralocorticoid receptor activation by cortisol and hypokalemic low renin hypertension as the main clinical feature. OBJECTIVE The objective of the study was to characterize AME for possible novel HSD11B2 mutations and to define the role of HSD11B2 promoter methylation in the phenotypic expression of the disease. SUBJECTS Two proband brothers and 10 relatives participated in the study. METHODS Peripheral blood mononuclear cell DNA was used for HSD11B2 exon sequencing, and a new predicted structure of 11β-hydroxysteroid dehydrogenase type 2 was generated by an in silico three-dimensional modeling. Promoter methylation was determined by bisulfite pyrosequencing. Urinary tetrahydrocortisol plus allotetrahydrocortisol to tetrahydrocortisone ratio, a surrogate marker of 11β-hydroxysteroid dehydrogenase type 2 activity, was measured by gas chromatography-mass spectrometry. RESULTS A novel homozygous variant at HSD11B2 exon 3 site (c.C662G) resulting in an alanine-to-glycine change at position 221 was discovered by sequencing the DNA of the probands. A monoallelic mutation was found in the DNA of the parents and other four relatives. In silico three-dimensional modeling showed that the Ala221Gly substitution could perturb a hydrophobic interaction by reducing the enzymatic affinity for the substrate. The HSD11B2 promoter methylation of normotensive heterozygous relatives was similar to that of wild types, whereas the hypertensive heterozygous subjects showed higher methylation than wild types, consistently with a transcriptional repressive effect of promoter hypermethylation. CONCLUSIONS A novel HSD11B2 functional mutation accounting for an Ala221Gly substitution causes AME. The hypertension phenotype is also epigenetically modulated by HSD11B2 methylation in subjects heterozygous for the mutation.

Circadian exosomal expression of renal thiazide-sensitive NaCl cotransporter (NCC) and prostasin in healthy individuals

A circadian timing system is involved in the maintenance of fluid and electrolyte balance and blood pressure control. Aldosterone and vasopressin modulate ion transporters and channels crucial in sodium (Na) and water reabsorption such as the epithelium Na channel and the renal thiazide‐sensitive NaCl cotransporter (NCC). We analyzed in urinary exosomes the intraday variations of NCC and prostasin expression and the association with electrolytes and water balance parameters.

Increased urinary excretion of the epithelial Na channel activator prostasin in patients with primary aldosteronism

Objectives: Prostasin is a glycosylphosphatidylinositol-anchored serine protease that is released in urine and is involved in epithelial Na channel activation. A direct association between urinary prostasin (u-prostasin) concentration and activation of the aldosterone-driven pathway has been suggested; however, in previous studies on primary aldosteronism, a semiquantitative evaluation, rather than a precise quantification, of prostasin was performed. We aim to investigate if u-prostasin concentrations are higher in patients with primary aldosteronism than in patients with essential hypertension and whether u-prostasin measurements could be a useful marker for diagnosing primary aldosteronism in hypertensive patients. Methods: A total of 62 primary aldosteronism and 56 essential hypertension patients were enrolled. Biochemical and hormonal parameters were measured by applying routine laboratory methods, and u-prostasin levels were assessed by ELISA. Results: Primary aldosteronism patients had higher u-prostasin levels than did essential hypertension patients. Prostasin levels were positively correlated with the aldosterone-to-renin ratio and inversely correlated with plasma K and urinary Na levels. In the highest concentration quartile, u-prostasin levels were associated with a several-fold higher probability of primary aldosteronism diagnosis in hypertensive patients. Receiver operating characteristic curve analysis showed that prostasin was specific but poorly sensitive as a diagnostic marker for primary aldosteronism. Conclusions: The study shows that an elevated u-prostasin concentration in humans is a specific marker for primary aldosteronism, which involves the classical model of epithelial Na channel activation. There was no statistically significant difference in prostasin concentrations among patients with different primary aldosteronism subtypes. Studies with a larger series of patients are necessary to clarify the clinical usefulness of the prostasin assay.

Urinary protease inhibitor Serpin B3 is higher in women and is further increased in female patients affected by aldosterone producing adenoma.

A correct diagnosis of primary aldosteronism (PA) requires adrenal venous sampling (AVS) for the classification of subtypes (bilateral hyperplasia, BAH, or adenoma, APA). Since such testing is not easily practicable, appropriate markers for the definition of subtypes are desirable. We hypothesized that an aldosterone excess was associated with abnormalities in urinary proteome, specific for PA subtypes. The project work was divided into 3 phases: (1) screening/identification by proteomic analysis and further characterization by RT-PCR and immunohistochemistry of the candidate protein; (2) clinical validation by quantitative ELISA assay of 57 (33 M, 24 F) PA patients and 50 normotensive controls (21 M, 29 F); (3) analysis of adrenal tissue of 8 individuals who had undergone adrenalectomy for APA or other adrenal tumors. The proteomic analysis showed a different expression of Serpin B3 Inhibitor-SCCA1 (SB3) in APA and BAH patients. Urine SB3 concentrations in normotensive controls, quantified by ELISA assay and normalized by urinary creatinine, resulted much lower in males (6.72 ng SB3 per mg creatinine, C.I. 4.43-10.19) than in females (20.56 ng SB3 per mg creatinine, C.I. 12.43-33.99, p < 0.00001). SB3 concentrations were not significantly different in males affected by different PA subtypes (BAH, n = 19 and APA, n = 14) compared with normotensive subjects (n = 21). In contrast, in PA females, SB3 was significantly higher in APA (n = 13) than in BAH patients (n = 11) or in normotensive controls (n = 29) (P < 0.01 and <0.05, respectively). Neither messenger RNA nor SB3 protein were identified in tissue obtained from adrenal tumors and from the surrounding normal gland. In conclusion urine SB3 concentrations are physiologically much lower in males than in females. Hypertensive women, affected by APA, present urinary SB3 concentrations significantly higher than women affected by BAH.

[LB.01.32] URINARY METABOLIC SIGNATURE OF PRIMARY ALDOSTERONISM: GENDER AND SUBTYPE SPECIFIC ALTERATIONS

Objective: Background. Current work up for primary aldosteronism diagnosis requires, from the initial suspicious to the final subtype classification, complex and expensive tests which include invasive approaches performed by highly skilled personnel; appropriate markers for the definition of subtype are thus greatly desirable. Recently we demonstrated that an aldosterone excess was associated with subtype-specific features in urinary proteome. Design and method: Aims and Methods. We performed a mass spectrometry-based metabolomics analysis looking for specific urinary molecular signatures of primary aldosteronism. We studied patients affected by essential hypertension and primary aldosteronism and matched-healthy subjects. The study was planned to take into account gender-related differences, while adopting advanced UHPLC-MS metabolomics analyses. Results: Results. As general results, we recognized statistically significant changes (p < 0.05 ANOVA, Fc > 1.5) of metabolites involved in central carbon, energy and nitrogen metabolism, especially purine and pyrimidine nucleosides and precursors, and free amino acids. Partial least squares discriminant analysis interpretation provided strong evidence about a disease-specific metabolic pattern with dAMP, diiodothyronine and 5-methoxytryptophan as leading factors, and a sex-specific metabolic pattern, associated with orotidine 5-phosphate, N-acetylalanine, hydroxyproline and cysteine. Conclusions: Conclusion. Despite the exploratory nature of this study, preliminary results highlight for the first time specific urinary metabolic signatures that can discriminate gender- and PA-subtype phenotypes.