Giraffe’s urine: from urinalysis to proteomics and metabolomics

This thesis focused on defining the urinary reference values and investigating the urinary proteome and metabolome of captive giraffes (Giraffa camelopardalis) by using a non-invasive sampling method. Urine samples were collected with a syringe from the ground, immediately after spontaneous voiding, by aspirating the upper part of samples, to avoid them to come into contact with the soil contaminants as much as possible. To prove the sampling’s reliability, cow urines were used and the results of both types of samples (those collected in sterile urine cups and those collected from the ground with a syringe) were compared. This experiment revealed no statistical differences in the variables investigated (urine total protein, uTP; urine creatinine, uCrea; urine protein:creatinine ratio, UPC; number of protein bands and band protein quantification detected by 1D-electrophoresis-SDS-PAGE), which proved the reliability of this sampling technique. By establishing the urinary reference values as well as by identifying the urinary proteome, it was possible to obtain information about the renal function in giraffes for the first time. Just like other ruminants, urine of giraffes contains low quantity of proteins, the majority of which shows low molecular mass (MM). The proteins identified in their urines might act as a defence against microbes and play a role in the ability of giraffes to concentrate urine. A first insight into the urinary metabolome allowed to identify and quantify 39 molecules; this provided some information on some physiological adaptations of giraffes and were influenced by sex and age

Onconase Restores Cytotoxicity in Dabrafenib-Resistant A375 Human Melanoma Cells and Affects Cell Migration, Invasion and Colony Formation Capability

Melanoma is a lethal tumor because of its severe metastatic potential, and serine/threonine-protein kinase B-raf inhibitors (BRAFi) are used in patients harboring BRAF-mutation. Unfortunately, BRAFi induce resistance. Therefore, we tested the activity of onconase (ONC), a cytotoxic RNase variant, against BRAFi-resistant cells to re-establish the efficacy of the chemotherapy. To do so, an A375 dabrafenib-resistant (A375DR) melanoma cell subpopulation was selected and its behavior compared with that of parental (A375P) cells by crystal violet, 5-Bromo-2'-deoxyuridine incorporation, and cleaved poly(ADP-ribose) polymerase 1 (PARP1) western blot measurements. Then, nuclear p65 Nuclear Factor kappaB (NF-\u3baB) and I\u3baB kinases-\u3b1/\u3b2 (IKK) phosphorylation levels were measured. Gelatin zymography was performed to evaluate metalloproteinase 2 (MMP2) activity. In addition, assays to measure migration, invasion and soft agar colony formation were performed to examine the tumor cell dissemination propensity. ONC affected the total viability and the proliferation rate of both A375P and A375DR cell subpopulations in a dose-dependent manner and also induced apoptotic cell death. Among its pleiotropic effects, ONC reduced nuclear p65 NF-\u3baB amount and IKK phosphorylation level, as well as MMP2 activity in both cell subpopulations. ONC decreased cell colony formation, migration, and invasion capability. Notably, it induced apoptosis and inhibited colony formation and invasiveness more extensively in A375DR than in A375P cells. In conclusion, ONC successfully counteracts melanoma malignancy especially in BRAFi-resistant cells and could become a tool against melanoma recurrence

Dimerization of human angiogenin and of variants involved in neurodegenerative diseases

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies

NMR characterization of angiogenin variants and tRNAAla products impacting aberrant protein oligomerization

Protein oligomerization is key to countless physiological processes, but also to abnormal amyloid conformations implicated in over 25 mortal human diseases. Human Angiogenin (h-ANG), a ribonuclease A family member, produces RNA fragments that regulate ribosome formation, the creation of new blood vessels and stress granule function. Too little h-ANG activity leads to abnormal protein oligomerization, resulting in Amyotrophic Lateral Sclerosis (ALS) or Parkinson's disease. While a score of disease linked h-ANG mutants has been studied by X-ray diffraction, some elude crystallization. There is also a debate regarding the structure that RNA fragments adopt after cleavage by h-ANG. Here, to better understand the beginning of the process that leads to aberrant protein oligomerization, the solution secondary structure and residue-level dynamics of WT h-ANG and two mutants i.e., H13A and R121C, are characterized by multidimensional heteronuclear NMR spectroscopy under near-physiological conditions. All three variants are found to adopt well folded and highly rigid structures in the solution, although the elements of secondary structure are somewhat shorter than those observed in crystallography studies. R121C alters the environment of nearby residues only. By contrast, the mutation H13A affects local residues as well as nearby active site residues K40 and H114. The conformation characterization by CD and 1D 1H NMR spectroscopies of tRNAAla before and after h-ANG cleavage reveals a retention of the duplex structure and little or no G-quadruplex formation

RNase A domain-swapped dimers produced through different methods: structure\u2013catalytic properties and antitumor activity

Upon oligomerization, RNase A can acquire important properties, such as cytotoxicity against leukemic cells. When lyophilized from 40% acetic acid solutions, the enzyme self-associates through the so-called three-dimensional domain swapping (3D-DS) mechanism involving both N- and/or C-terminals. The same species are formed if the enzyme is subjected to thermal incubation in various solvents, especially in 40% ethanol. We evaluated here if significant structural modifications might occur in RNase A N- or C-swapped dimers and/or in the residual monomer(s), as a function of the oligomerization protocol applied. We detected that the monomer activity vs. ss-RNA was partly affected by both protocols, although the protein does not suffer spectroscopic alterations. Instead, the two N-swapped dimers showed differences in the fluorescence emission spectra but almost identical enzymatic activities, while the C-swapped dimers displayed slightly different activities vs. both ss- or ds-RNA substrates together with not negligible fluorescence emission alterations within each other. Besides these results, we also discuss the reasons justifying the different relative enzymatic activities displayed by the N-dimers and C-dimers. Last, similarly with data previously registered in a mouse model, we found that both dimeric species significantly decrease human melanoma A375 cell viability, while only N-dimers reduce human melanoma MeWo cell growth

“Urinalysis, urinary proteome and metabolome of zoo-housed giraffes (Giraffa camelopardalis) through noninvasive sampling method”.

The study of non-domestic animals withholds more difficulties compared to the domestic counterpart, thus using noninvasive techniques to collect biological samples might play an important role in assessing the health status of wild animals. 1 The present study established the reliability of urine sampling from the ground. A preliminary study was run with 10 urine samples of 10 cows (Bos taurus) housed in a dairy farming in Northern Italy. Urine samples, collected both in sterile cups and from the ground, were analyzed and compared. Results revealed no statistical differences in the variables investigated (p > 0.05, dipstick parameters and USG, protein quantification and UPC and protein electrophoresis), which proved the reliability of this noninvasive sampling method. This method was used for sampling 103 urine samples from 44 zoo-housed giraffes (Giraffa camelopardalis) of four Italian zoos. Urine samples were used to establish the urinalysis reference values in this species and to study the urinary proteome for the first time. The urinary reference values reported as median (lower and upper limit) were: urine specific gravity (USG), 1.030 (1006 - 1.049); urine total proteins (uTP), 17.58 (4.54 – 35.31) mg/dL; urine creatinine (uCr), 154.62 (39.59 – 357.95) mg/dL; urine protein: creatinine ratio (UPC), 0.11 (0.07 – 0.16). In giraffes, most urinary proteins had a low molecular mass (MM) and were present in low quantities. Proteomics disclosed fifteen different proteins, which were involved in the defense against microbes and in the ability of giraffes to concentrate urine. Albumin, lysozyme C, and ubiquitin were the most represented urinary proteins in giraffes. In addition, to define the urinary metabolome profile, 35 urine samples from 35 zoo-housed giraffes of five Italian zoos were used. Metabolomics allowed to identify and quantify 39 molecules and the most represented metabolites were hippurate, creatinine and phenylacetylglycine. This analysis provided information on physiological adaptations of giraffes. Besides, urinary metabolites were influenced by sex: urinary metabolome profile of female showed higher level of acetate, succinate, and lactate, conversely hippurate, phenylacetylglycine, and thymine were more concentrated in male urines. Similarly, the age affected the concentration of three urinary metabolites, namely formate, alanine, and valerate

STRUCTURAL DETERMINANTS AFFECTING THE OLIGOMERIZATION TENDENCY OF SOME PANCREATIC RIBONUCLEASES

The three dimensional domain swapping (3D-DS) mechanism represents a useful strategy that proteins can follow to self-associate. This mechanism requires the presence of a flexible portion that generally links the N- and/or C-terminus of the protein to its core. This portion, called hinge loop, can adopt different conformations as a function of the environmental conditions and allows the mentioned N- and/or C-terminal domains to be detached from the protein core and to be swapped with an identical domain of another protomer. This induces the formation of dimer(s) or larger oligomer(s) that often display, or enforce, biological properties that are absent, or attenuated, in the native monomer. The 3D-DS mechanism is shared by some proteins involved in amyloidosis, such as the human prion protein, \u3b22-microglobulin, or some cystatins. Although not being amyloidogenic, the \u201cpancreatic-type\u201d RNases, i.e. resembling the features of the well-known pancreatic bovine RNase A, have become milestones to comprehend the determinants ruling out the 3D-DS mechanism. This is true especially for RNase A and for its natively dimeric homolog bovine seminal (BS)-RNase, whose structural determinants characterizing their self-association through 3D-DS have been deeply studied in the recent past. Moreover, also other ribonucleases included in the same RNase A super-family can oligomerize through this mechanism, as for example onconase (ONC), that can be induced to dimerize through the 3D-DS of its N-terminal domain. In this thesis, my aim was to elucidate some structural determinants that settle the tendency of some pancreatic-type RNases to self-associate, keeping the known features of RNase A as a reference. Therefore, we firstly investigated if the different methods that can be used to obtain the oligomers of RNase A might influence their properties. We treated RNase A with two different protocols: in particular, i. lyophilization of acetic acid solutions of the protein, or, ii. thermal incubations performed at 60\ub0C in 20 or 40% aqueous ethanol. We observed that the enzymatic activity of the monomers and of the N- and C-swapped dimers (ND and CD, respectively) are slightly affected by the particular method used to induce their self-association. Then, I focused my attention to onconase (ONC), an amphibian member of the \u201cpancreatic-type\u201d RNase super-family lead by RNase A. In particular my aim was to unlock its C-terminal domain, that in the wild-type is blocked by a disulfide bond involving its C-terminal Cys104 residue, in order to investigate if this RNase variant may dimerize also through the C-terminal-end swapping. Unfortunately, none of numerous ONC mutant that I produced could form either the C-dimer, or also larger oligomers. In addition, all these variants formed less N-dimer than the wild type, confirming the actual mutual influence existing between the N- and C-termini of the protein, as it occurs for other RNases. We also supposed that the impossibility for ONC to form a C-dimer could be ascribable to the elongation of its C-terminal domain, with respect to RNase A. Therefore, we analyzed also the aggregation propensity of human pancreatic RNase, a variant that displays a C-terminus elongation of four AA residues in comparison with RNase A, similarly to ONC. Both the wt and a mutant obtained by the deleting the four mentioned residues displayed SEC profiles qualitatively very similar to the one of RNase A. Therefore, the human variant actually displayed that it can extensively oligomerize, but we could also deduce that the C-terminal elongation influences only marginally this process. The last project I developed was focused on the analysis of the oligomerization tendency of human angiogenin (ANG), a 14 kDa RNase variant characterized by a low ribonucleolytic activity (10-5/10-6 fold less than of RNase A), however necessary for its crucial angiogenic effects. ANG is involved in tumorigenesis but it exerts also a survival-promoting effect on the central nervous system (CNS) neuronal progenitors. However, some ANG variants are involved in neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) and Parkinson Disease (PD). ALS is a multifactor disease, but one hypothesis to explain the pathogenic effect could be related to a possible oligomerization and precipitation of the mutants in the CNS. Among the numerous pathogenic ANG variants existing, one candidate retained by some scientists prone to undergo self-association is the S28N mutant. In our hands, this variant showed to dimerize at a slightly higher extent than the wild type, that in turn dimerized as well. Then, other ANG pathogenic mutants, in particular H13A and Q117G, also showed to dimerize, but with less reproducible results. Anyway, we detected for the first time that also ANG can dimerize through the 3D-DS mechanism, as many other RNases do. In conclusion, all the results of this thesis can be considered a step forward to comprehend the determinants settling the 3D-DS dimerization, or oligomerization tendency of many pancreatic-type RNases, and to compare the determinants that induce the differences emerging within them

Dimerization of Human Angiogenin and of Variants Involved in Neurodegenerative Diseases

Human Angiogenin (hANG, or ANG, 14.1 kDa) promotes vessel formation and is also called RNase 5 because it is included in the pancreatic-type ribonuclease (pt-RNase) super-family. Although low, its ribonucleolytic activity is crucial for angiogenesis in tumor tissues but also in the physiological development of the Central Nervous System (CNS) neuronal progenitors. Nevertheless, some ANG variants are involved in both neurodegenerative Parkinson disease (PD) and Amyotrophic Lateral Sclerosis (ALS). Notably, some pt-RNases acquire new biological functions upon oligomerization. Considering neurodegenerative diseases correlation with massive protein aggregation, we analyzed the aggregation propensity of ANG and of three of its pathogenic variants, namely H13A, S28N, and R121C. We found no massive aggregation, but wt-ANG, as well as S28N and R121C variants, can form an enzymatically active dimer, which is called ANG-D. By contrast, the enzymatically inactive H13A-ANG does not dimerize. Corroborated by a specific cross-linking analysis and by the behavior of H13A-ANG that in turn lacks one of the two His active site residues necessary for pt-RNases to self-associate through the three-dimensional domain swapping (3D-DS), we demonstrate that ANG actually dimerizes through 3D-DS. Then, we deduce by size exclusion chromatography (SEC) and modeling that ANG-D forms through the swapping of ANG N-termini. In light of these novelties, we can expect future investigations to unveil other ANG determinants possibly related with the onset and/or development of neurodegenerative pathologies

First Insights into the Urinary Metabolome of Captive Giraffes by Proton Nuclear Magnetic Resonance Spectroscopy

The urine from 35 giraffes was studied by untargeted 1H-NMR, with the purpose of obtaining, for the first time, a fingerprint of its metabolome. The metabolome, as downstream of the transcriptome and proteome, has been considered as the most representative approach to monitor the relationships between animal physiological features and environment. Thirty-nine molecules were unambiguously quantified, able to give information about diet, proteins digestion, energy generation, and gut-microbial co-metabolism. The samples collected allowed study of the effects of age and sex on the giraffe urinary metabolome. In addition, preliminary information about how sampling procedure and pregnancy could affect a giraffe’s urinary metabolome was obtained. Such work could trigger the setting up of methods to non-invasively study the health status of giraffes, which is utterly needed, considering that anesthetic-related complications make their immobilization a very risky practice