Papers by Massimiliano Gaetani
Multifaceted interrogation of the proteome deepens the system-wide understanding of biological sy... more Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, we devise the first high- throughput redox proteomics approach integrated with expression analysis (REX) and combine it with Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, we analyzed HCT116 cells treated with auranofin, showing great improvement compared with previous such studies. Then we applied PISA-REX to study proteome remodeling upon stimulation of human monocytes by interferon α. We also studied the proteome changes in plasmacytoid dendritic cells isolated from wild type vs.Ncf1- mutant mice treated with interferon α, showing that NCF1 deficiency enhan...
Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physi... more Inorganic materials depleted of heavy stable isotopes are known to deviate strongly in some physico-chemical properties from their isotopically natural (native) counterparts; however, in biotechnology such effects have not been investigated yet. Here we explored for the first time the effect of simultaneous depletion of the heavy carbon, hydrogen, oxygen and nitrogen isotopes on the bacterium E. coli and the enzymes expressed in it. Bacteria showed faster growth, with proteins exhibiting higher thermal stability, while for recombinant enzymes expressed in ultralight media, faster kinetics was discovered. At room temperature, luciferase, thioredoxin and dihydrofolate reductase showed a 40-250% increase in activity compared to the native counterparts. The efficiency of ultralight Pfu DNA polymerase in polymerase chain reaction was also significantly higher than that of the normal enzyme. At 10 °C, the advantage factor of ultralight enzymes typically increased by 50%, which points towa...
ABSTRACTMutations in hundreds of genes have been associated with formation of human cancer, with ... more ABSTRACTMutations in hundreds of genes have been associated with formation of human cancer, with different oncogenic lesions prevalent in different cancer types. Yet, the malignant phenotype is simple, characterized by unrestricted growth of cells that invade neighboring healthy tissue and in many cases metastasize to distant organs. One possible hypothesis explaining this dichotomy is that the cancer genes regulate a common set of target genes, which then function as master regulators of essential cancer phenotypes, such as growth, invasion and metastasis. To identify mechanisms that drive the most fundamental feature shared by all tumors – unrestricted cell proliferation – we used a multiomic approach to identify common transcriptional and posttranslational targets of major oncogenic pathways active in different cancer types, and combined this analysis with known regulators of the cell cycle. We identified translation and ribosome biogenesis as common targets of both transcription...
Viruses
Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and ... more Natural killer (NK) cells mount an immune response against hepatitis C virus (HCV) infection and can be activated by several cytokines, including interleukin-2 (IL-2), IL-15, and interferon-alpha (IFN-α). By exploiting the Huh7.5 hepatoma cell line infected with the HCV JFH1 genome, we provide novel insights into the antiviral effector functions of human primary NK cells after cytokine stimulation. NK cells activated with IFN-α (IFNα-NKs) had enhanced contact-dependent and -independent responses as compared with NK cells activated with IL-2/IL-15 (IL2/IL15-NKs) and could inhibit HCV replication both in vitro and in vivo. Importantly, IFN-α, but not IL-2/IL-15, protected NK cells from the functional inhibition exerted by HCV. By performing flow cytometry, multiplex cytokine profiling, and mass-spectrometry-based proteomics, we discovered that IFNα-NKs secreted high levels of galectin-9 and interferon-gamma (IFN-γ), and by conducting neutralization assays, we confirmed the major role ...
Journal of Biological Chemistry, 2021
Human Reproduction, 2021
Study question What are the therapeutic targets and mechanisms of green tea EGCG and Pro-EGCG in ... more Study question What are the therapeutic targets and mechanisms of green tea EGCG and Pro-EGCG in treating endometriosis? Summary answer EGCG and Pro-EGCG have unique molecular targets to regulate interactions of B cells, macrophages and endometriotic cells and limit the growth and development of endometriosis. What is known already Current treatments of endometriosis are mainly hormonal suppression and surgical ablation or removal. Our previous studies showed EGCG significantly inhibits development of experimental endometriosis in mice. Pro-EGCG is more effective than EGCG in term of anti-endometriosis, anti-angiogenesis and anti-oxidation (Wang, et. al., 2013; Xu, et al., 2011). Dysfunctional immunological activities of macrophages and B cells were found in women with endometriosis. The molecular targets, underlying mechanism and differential therapeutic efficacy of EGCG and Pro-EGCG, as well as their anti-inflammatory activities are still not known. Study design, size, duration Mu...
Nature Communications, 2021
Detailed characterization of cell type transitions is essential for cell biology in general and p... more Detailed characterization of cell type transitions is essential for cell biology in general and particularly for the development of stem cell-based therapies in regenerative medicine. To systematically study such transitions, we introduce a method that simultaneously measures protein expression and thermal stability changes in cells and provide the web-based visualization tool ProteoTracker. We apply our method to study differences between human pluripotent stem cells and several cell types including their parental cell line and differentiated progeny. We detect alterations of protein properties in numerous cellular pathways and components including ribosome biogenesis and demonstrate that modulation of ribosome maturation through SBDS protein can be helpful for manipulating cell stemness in vitro. Using our integrative proteomics approach and the web-based tool, we uncover a molecular basis for the uncoupling of robust transcription from parsimonious translation in stem cells and p...
Enzyme and Microbial Technology, 2005
As the final step of a study aiming at the optimization of culture conditions for the production ... more As the final step of a study aiming at the optimization of culture conditions for the production of carotenoids by red yeasts, a statisticallybased experimental design has been applied to assess the influence of selected trace elements on carotenogenesis in Rhodotorula graminis DBVPG 7021. In particular, a central composite design scheme has been used to evaluate the influence of Fe 3+ , Co 2+ , Mn 2+ , Al 2+ and Zn 2+ (within the range 0-50 ppm) on various responses, namely biomass (B), total carotenoid production (TC) and percentage of specific carotenoids (-CAR, -carotene; ␥-CAR, ␥-carotene; TN, torulene; TD, torularhodin) on total carotenoids. Second-order polynomial models were calculated and reduced equations were designed by neglecting non-significant (P < 0.01) regression coefficients. Reduced equations were used to calculate the optimal concentration of trace elements in view of maximising the level of B, TC, -CAR, ␥-CAR, TN and TD. After optimization, average final values total carotenoids (TC = 803.2 g/g DW) was about 370% of value observed as central point of the central composite design scheme. Under the same condition, average final values of other responses were: B = 5.40 g/L; -CAR = 50.3%; ␥-CAR = 15.4%; TN = 22.7%; TD = 11.6%. All above experimental data are in good agreement with calculated ones, thus confirming the reliability of the proposed empirical model in describing carotenoid production by R. graminis as a function of trace element concentrations.
O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyz... more O-GalNAc type glycosylation is a common post-translational modification (PTM) of proteins catalyzed by polypeptide GalNAc transferases, but the substrate specificity of these transferases is poorly understood. Here we develop a strategy based on integral thermal proteome solubility profiling to identify and prioritize the protein substrates of polypeptide N-acetylgalactosaminyltransferase 1 (GALNT1). Combined with glycoprotein enrichment followed by HCD and soft EThcD gas-phase fragmentation technique, we uncover hundreds of novel GALNT1 substrates in two model human cell lines. GALNT1-mediated O-glycosylation is more common on Thr than Ser residues, with a strong preference for Pro at positions +3 and +4 in respect to O-glycosylation. These results implicate GALNT1 in potentially regulating proteins in several diverse pathways, including some unexpected processes, such as TCA cycle and DNA transcription. This study depicts a roadmap for identification of functional substrates for g...
21A is an <i>Alu</i> non-coding (nc) RNA transcribed by RNA polymerase (pol) III. Whi... more 21A is an <i>Alu</i> non-coding (nc) RNA transcribed by RNA polymerase (pol) III. While investigating the biological role of 21A ncRNA we documented an inverse correlation between its expression level and the rate of cell proliferation. The down-regulation of this ncRNA not only caused a boost in cell proliferation, but was also associated to a transient cell dedifferentiation, suggesting a possible involvement of this RNA in cell dedifferentiation/reprogramming. In this study, we explored the possibility to enhance proliferation and dedifferentiation of cells of interest, by 21A down-regulation, using a mixture of chemically modified Anti-21A RNAs. Our results confirmed the validity of this approach that could allow the amplification of specific cell populations, in a controlled manner and without inducing permanent effects. In addition to induce cell proliferation, the procedure did not decrease the tissue regeneration potential of progenitor cells in two different cel...
Cells
The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel... more The in vivo-relevant phenotype of 3D liver spheroids allows for long-term studies of, e.g., novel mechanisms of chronic drug-induced liver toxicity. Using this system, we present a novel drug-induced stress response in human and murine hepatocyte spheroids, wherein long slender filaments form after chronic treatment with four different drugs, of which three are PPARα antagonists. The morphology of the thorns varies between donors and the compounds used. They are mainly composed of diverse protein fibres, which are glycosylated. Their formation is inhibited by treatment with fatty acids or antioxidants. Treatment of mice with GW6471 revealed changes in gene and protein expression, such as those in the spheroids. In addition, similar changes in keratin expression were seen following the treatment of hepatotoxic drugs, including aflatoxin B1, paracetamol, chlorpromazine, cyclosporine, and ketoconazole. We suggest that thorn formation may be indicative of hepatocyte metaplasia in respon...
Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal domina... more Mutations in PKD1, the gene encoding for the receptor Polycystin-1 (PC-1), cause autosomal dominant polycystic kidney disease (ADPKD). The cytoplasmic C-terminus of PC-1 contains a coiled-coil domain that mediates an interaction with the PKD2 gene product, Polycystin-2 (PC-2). Here we identify a novel domain in the PC-1 C-terminal tail, a polyproline motif mediating an interaction with Src homology domain 3 (SH3). A screen for interactions using the PC-1 C-terminal tail identified the SH3 domain of nephrocystin-1 (NPHP1) as a potential binding partner of PC-1. NPHP1 is the product of a gene that is mutated in a different form of renal cystic disease, nephronophthisis (NPHP). We show that in vitro pull-down assays and NMR structural studies confirmed the interaction between the PC-1 polyproline motif and the NPHP1 SH3 domain. Furthermore, the two full-length proteins interact through these domains; using a recently generated model system allowing us to track endogenous PC-1, we confi...
Pathogenic mutations in ␣ and  spectrin result in a variety of syndromes, including hereditary e... more Pathogenic mutations in ␣ and  spectrin result in a variety of syndromes, including hereditary elliptocytosis (HE), hereditary pyropoikilocytosis (HPP), and hereditary spherocytosis (HS). Although some mutations clearly lie at sites of interaction, such as the sites of spectrin ␣-tetramer formation, a surprising number of HE-causing mutations have been identified within linker regions between distal spectrin repeats. Here we apply solution structural and single molecule methods to the folding and stability of recombinant proteins consisting of the first 5 spectrin repeats of ␣-spectrin, comparing normal spectrin with a pathogenic linker mutation, Q471P, between repeats R4 and R5. Results show that the linker mutation destabilizes a significant fraction of the 5-repeat construct at 37°C, whereas the WT remains fully folded well above body temperature. In WT protein, helical linkers propagate stability from one repeat to the next, but the mutation disrupts the stabilizing influence of adjacent repeats. The results suggest a molecular mechanism for the high frequency of disease caused by proline mutations in spectrin linkers.
Experimental Hematology, 2020
Numerous strategies have been proposed to facilitate the ex vivo expansion of human hematopoietic... more Numerous strategies have been proposed to facilitate the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs) for clinical benefit using combinations of growth factors and small molecule compounds. Notably, UM171, a pyrimidoindole derivative, was shown to significantly expand the number of umbilical cord blood (UCB) HSPCs, and currently UM171 expanded UCB HSPCs are being tested in clinical trials with promising initial results. However, the precise target of UM171, and the molecular mechanisms of its function, have not yet been reported. We have found that inhibition of the epigenetic regulator Lysine-specific histone demethylase 1A (LSD1) induces a rapid expansion of human cord blood derived CD34+ cells and promotes in vitro propagation of long-term repopulating HSCs by preventing differentiation. Early transcriptional changes triggered by LSD1 inhibition in HSCs were strikingly similar to the gene signature induced upon UM171 treatment. To further explore thi...
• The common HE mutation aL260P reduces spectrin tetramer links between junctional complexes in r... more • The common HE mutation aL260P reduces spectrin tetramer links between junctional complexes in red cell membranes by favoring closed dimers. • Favoring closed spectrin dimer formation is a new mechanism of red cell membrane destabilization by hereditary anemia mutations. Hereditary elliptocytosis (HE) and hereditary pyropoikilocytosis (HPP) are common disorders of erythrocyte shape primarily because of mutations in spectrin. The most common HE/HPP mutations are located distant from the critical ab-spectrin tetramerization site, yet still interfere with formation of spectrin tetramers and destabilize the membrane by unknownmechanisms. To address this question, we studied the common HE-associated mutation, aL260P, in the context of a fully functional mini-spectrin. The mutation exhibited wild-type tetramer binding in univalent binding assays, but reduced binding affinity in bivalent-binding assays. Biophysical analyses demonstrated the mutation-containing domain was only modestly structurally destabilized and helical content was not significantly changed. Gel filtration analysis of the aL260P mini-spectrin indicated more compact structures for dimers and tetramers compared with wild-type. Chemical crosslinking showed structural changes in the mutant mini-spectrin dimer were primarily restricted to the vicinity of the aL260P mutation and indicated large conformational rearrangements of this region. These data indicate the mutation increased the stability of the closed dimer state, thereby reducing tetramer assembly and resulting in membrane destabilization. These results reveal a novel mechanism of erythrocyte membrane destabilization that could contribute to development of therapeutic interventions for mutations in membrane proteins containing spectrin-type domains associated with inherited disease. (Blood. 2013;122(17):3045-3053)
This protocol describes the proteomics technique called System-wide Identification and prioritiza... more This protocol describes the proteomics technique called System-wide Identification and prioritization of Enzyme Substrates by Thermal Analysis or SIESTA 1,2. SIESTA can be used for universal discovery of enzyme substrates that shift in thermal stability or solubility upon post-translational modification (PTM). Experimental design, proteomics sample preparation and data analysis are the key stages of this protocol. Data analysis can be performed using our SIESTA package hosted on GitHub 3. When performed with classical thermal proteome profiling (TPP), the protocol will take 5 days for sample preparation and 14 days of sample analysis by mass spectrometry (the current protocol). If our high-throughput version of TPP called Proteome Integral Solubility Alteration assay (PISA) 4 is used instead, the sample analysis time by mass spectrometry is reduced to 1-2 days for the same number of conditions.
The road to development of a new drug often begins with the idea of its target protein, which cou... more The road to development of a new drug often begins with the idea of its target protein, which could result from evidence emerging from different research sources, including academia, clinics and industry. However, taking a single compound to the drug market involves a very high cost, often over a billion of US dollars, and requires on average a time commitment between ten and fifteen years. The entire process is very complicated and, even in the lucky cases when the final development steps are reached, it sometimes collapses because of a failure related to the drug target, which is its starting point. Indeed, one of the major reasons of drug failures in clinical trials is the lack of efficacy due to low specificity of the drug towards the desired target. Sometimes, when the promising candidate compound is found in a large-scale phenotypic screening, the target is not even known a priori. Thus both identification and validation of targets are fundamental processes, and major bottlene...
It has been known from the earliest days of two-dimensional NMR that in principle a complete sepa... more It has been known from the earliest days of two-dimensional NMR that in principle a complete separation of proton shifts and proton-proton couplings can be achieved by recording a two-dimensional J-spectrum and then tilting this by 45°. However, the difficulty with such spectra is that the lines are phase-twists. Due to the dispersive component of this lineshape the resolution of the spectra are severely compromised. In addition, the 45° projection of the spectrum, which gives a proton-decoupled proton spectrum, is zero. Over the years, much effort has been put into trying to get around this problem, principally by eliminating the phase-twist lineshape; none of these methods seem to have gained wide acceptance. Recently we have developed two new experimental methods which, in effect, result in two-dimensional J-spectra with absorption mode lineshapes and which also preserve the natural intensities. The first [1] is based on an anti Z-COSY experiment, while the second [2] utilizes a novel pulse sequence element proposed by Zangger and Sterk [3]. These two methods will be described and possible applications outlined.
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Papers by Massimiliano Gaetani