Biology subjects

Tedesco, D.

Publications and source records attributed to Tedesco, D..

7 recordsLinked to original sources

bioRxiv2023

Western diet dampens T regulatory cell function to fuel hepatic inflammation in nonalcoholic fatty liver disease

Background and aimsThe immunosuppressive T regulatory cells (Tregs) regulate immune responses and maintain immune homeostasis, yet their functions in nonalcoholic fatty liver disease (NAFLD) pathogenesis remains controversial. MethodsMice were fed a normal diet (ND) or a western diet (WD) for 16 weeks to induce NAFLD. Diphtheria toxin injection to deplete Tregs in Foxp3DTR mice or Treg induction therapy in WT mice to augment Treg numbers was initiated at twelve and eight weeks, respectively. Liver tissues from mice and NASH human subjects were analyzed by histology, confocal imaging, and qRT-PCR. ResultsWD triggered accumulation of adaptive immune cells, including Tregs and effector T cells, within the liver parenchyma. This pattern was also observed in NASH patients, where an increase in intrahepatic Tregs was noted. In the absence of adaptive immune cells in Rag1 KO mice, WD promoted accumulation of intrahepatic neutrophils and macrophages and exacerbated hepatic inflammation and fibrosis. Similarly, targeted Treg depletion exacerbated WD-induced hepatic inflammation and fibrosis. In Treg-depleted mice, hepatic injury was associated with increased accumulation of neutrophils, macrophages, and activated T cells within the liver. Conversely, induction of Tregs using recombinant IL2/IL2 mAb cocktail reduced hepatic steatosis, inflammation, and fibrosis in WD-fed mice. Analysis of intrahepatic Tregs from WD-fed mice revealed a phenotypic signature of impaired Treg function in NAFLD. Ex vivo functional studies showed that glucose and palmitate, but not fructose, impaired the immunosuppressive ability of Treg cells. ConclusionsOur findings indicate that the liver microenvironment in NAFLD impairs ability of Tregs to suppress effector immune cell activation, thus perpetuating chronic inflammation and driving NAFLD progression. These data suggest that targeted approaches aimed at restoring Treg function may represent a potential therapeutic strategy for treating NAFLD. Lay summaryIn this study, we elucidate the mechanisms contributing to the perpetuation of chronic hepatic inflammation in nonalcoholic fatty liver disease (NAFLD). We show that dietary sugar and fatty acids promote chronic hepatic inflammation in NAFLD by impairing immunosuppressive function of regulatory T cells. Finally, our preclinical data suggest that targeted approaches aimed at restoring T regulatory cell function have the potential to treat NAFLD.

pathology↗

bioRxiv2022

Structural and functional characterization of chloroplast ribulose-5-phosphate-3-epimerase from the model green microalga Chlamydomonas reinhardtii

Photosynthetic carbon fixation relies on Rubisco and ten additional enzymes in the conserved Calvin-Benson-Bassham (CBB) cycle. Epimerization of xylulose-5-phosphate (X5P) into ribulose-5-phosphate (Ru5P) contributes to the regeneration of ribulose-1,5-bisphosphate, the substrate of Rubisco activity. Ribulose-5-phosphate-3-epimerase (RPE) catalyzes the formation of Ru5P but it can also operate in the pentose phosphate pathway (PPP) by catalyzing the reverse reaction. Here, we describe the catalytic and structural properties of the recombinant form of photosynthetic RPE isoform 1 from Chlamydomonas reinhardtii (CrRPE1). The enzyme shows catalytic parameters that are variably comparable to those of the paralogues involved in the PPP and CBB cycle but with some notable exceptions. CrRPE1 is a homo-hexamer that exposes a catalytic pocket on the top of an 8{beta}8 triose isomerase-type (TIM-) barrel as observed in structurally solved RPE isoforms from both plant and non-plant sources. Despite being identified as a putative target of thiol-based redox modifications, CrRPE1 activity is not altered by redox treatments, indicating that the enzyme does not bear redox sensitive thiol groups and is not regulated by thiol-switching mechanisms. We mapped phosphorylation sites on the crystal structure and the specific location at the entrance of the catalytic cleft supports a phosphorylation-based regulatory mechanism. Overall, this work provides a detailed description of the catalytic and regulatory properties of CrRPE along with structural data, which allow for a deeper understanding of the functioning of this enzyme of the CBB cycle and in setting the basis for possible strategies to improve the photosynthetic metabolism.

biochemistry↗

bioRxiv2021

Application of the SMALP technology to the isolation of GPCRs from low-yielding cell lines

The ability of styrene-maleic acid (SMAc) co-polymers to spontaneously insert into biological membranes can be exploited to extract G protein-coupled receptors (GPCRs) embedded in styrene-maleic acid lipid particles (SMALPs), preserving the native environment around the protein and thus enhancing the feasibility of functional studies. So far, the SMALP technology has been primarily employed on non-mammalian cells and protocols are not optimized for adherent human cell lines, which cannot be harvested in large amounts. In this work, a fine investigation of key parameters affecting the formation of SMALPs was undertaken with the purpose of maximizing the yield of extraction of a recombinant form of human {beta}2-adrenergic receptor (rh{beta}2AR) from HEK293T cells. The study highlighted an important influence of ionic strength on the membrane solubilization efficiency and GPCR purification yield of SMAc co-polymers: by lowering the salt concentration of all buffers used in previously published SMALP protocols, the water solubility and extraction efficiency of the selected SMAc co-polymer (commercially supplied as a potassium salt) were enhanced. In-line combination of size-exclusion chromatography (SEC) with immobilized metal affinity chromatography (IMAC) allowed further improvement of the final rh{beta}2AR yield by reducing the loss of SMALP-embedded GPCRs during the fractionation and purification of SMALPs. The overall findings of this study show that the available SMALP protocols can be significantly optimized in several aspects in order to increase the efficiency of GPCR solubilization and isolation from low-yielding expression systems. HighlightsO_LIThe SMALP technology enables a direct solubilization of GPCRs from cell membranes. C_LIO_LIThe isolation of GPCRs from mammalian cells is usually a low-yield procedure. C_LIO_LISMALPs embedding the {beta}2-adrenergic receptor were prepared from a HEK293T cell line. C_LIO_LIThe ionic strength of buffers plays a key role in SMALP formation and isolation. C_LIO_LIStandard SMALP protocols can be finely optimized to increase purification yields. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/428305v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@14a3bdcorg.highwire.dtl.DTLVardef@a00d14org.highwire.dtl.DTLVardef@1baca87org.highwire.dtl.DTLVardef@4686c7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Structural and functional insights into nitrosoglutathione reductase from Chlamydomonas reinhardtii

Protein S-nitrosylation plays a fundamental role in cell signaling and nitrosoglutathione (GSNO) is considered as the main nitrosylating signaling molecule. Enzymatic systems controlling GSNO homeostasis are thus crucial to indirectly control the formation of protein S-nitrosothiols. GSNO reductase (GSNOR) is the key enzyme controlling GSNO levels by catalyzing its degradation in the presence of NADH. Here, we found that protein extracts from the microalga Chlamydomonas reinhardtii catabolize GSNO via two enzymatic systems having specific reliance on NADPH or NADH and different biochemical features. Scoring the Chlamydomonas genome for orthologs of known plant GSNORs, we found two genes encoding for putative and almost identical GSNOR isoenzymes. One of the two, here named CrGSNOR1, was heterologously expressed and purified. The kinetic properties of CrGSNOR1 were determined and the high-resolution three-dimensional structures of the apo and NAD+-bound forms of the enzyme were solved. These analyses revealed that CrGSNOR1 has a strict specificity towards GSNO and NADH, and a conserved 3D-folding with respect to other plant GSNORs. The catalytic zinc ion, however, showed an unexpected variability of the coordination environment. Furthermore, we evaluated the catalytic response of CrGSNOR1 to thermal denaturation, thiol-modifying agents and oxidative modifications as well as the reactivity and position of accessible cysteines. Despite being a cysteine-rich protein, CrGSNOR1 contains only two solvent-exposed/reactive cysteines. Oxidizing and nitrosylating treatments have null or limited effects on CrGSNOR1 activity, highlighting a certain resistance of the algal enzyme to redox modifications. The molecular mechanisms and structural features underlying the response to thiol-based modifications are discussed. One-sentence summaryGSNOR1 from Chlamydomonas reinhardtii displays an unusual variability of the catalytic zinc coordination environment and an unexpected resistance to thiol-based redox modifications

plant biology↗

bioRxiv2020

Adaptation of STARR-seq method to be used with 3rd generation integrase-deficient promoterless lentiviral vectors

ABSTRACTAbility to functionally screen gene regulatory sequences, such as promoters and enhancers, in high throughput manner is an important prerequisite for many basic and translational research programs. One of the methods that allow such screening is STARR-seq, or self-transcribing active regulatory region sequencing. It allows to quickly screen millions of candidate sequences in the cell type of interest. However, it does rely on transfection as a delivery method which can be a limiting-step for some hard-to-transfect cells such as senescent cells. Here we show that integration-deficient and integration-competent promoterless lentiviral particles can be used to deliver STARR-seq constructs into cells. These constructs reported CMV enhancer activity both at protein and mRNA level. While further validations are necessary, ability to deliver STARR-seq libraries using lentiviral particles will significantly improve the versatility and usability of such a method.Competing Interest StatementThe authors have declared no competing interest.View Full Text

synthetic biology↗

bioRxiv2018

MafK mediates chromatin remodeling to silence IRF8 expression in non-immune cells in a lineage-specific manner

The regulation of gene expression is a result of a complex interplay between chromatin remodeling, transcription factors (TFs) and signaling molecules. Cell differentiation is accompanied by chromatin remodeling of specific loci to permanently silence genes that are not essential for the differentiated cell activity. The molecular cues that recruit the chromatin remodeling machinery are not well characterized. IRF8 is an immune-cell specific TF, and thus, serves as a model gene to elucidate the molecular mechanisms governing its silencing in non-immune cells. A high-throughput shRNA library screen in IRF8 expression-restrictive cells enabled the identification of MafK as modulator of IRF8 silencing, affecting chromatin architecture. ChIP-seq analysis revealed three MafK binding-regions (-25kb, -20kb and IRF8 6th intron) in the IRF8 locus. These MafK binding-sites are sufficient to repress a reporter-gene when cloned in genome-integrated lentiviral reporter constructs in expression-restrictive cells only. Conversely, plasmid-based constructs do not demonstrate such repressive effect. These results highlight the role of these MafK binding-sites in mediating repressed chromatin assembly. Furthermore, removal of MafK-int6 binding-region from BAC-IRF8 reporter construct was sufficient to promote accessible chromatin conformation. Taken together, we identified and characterized several MafK binding elements within the IRF8 locus that mediate repressive chromatin conformation resulting in the silencing of IRF8 expression only in non-immune cells.

molecular biology↗

bioRxiv2018

MDM4 is an essential disease driver targeted by 1q gain in Burkitt lymphoma

Oncogenic MYC activation promotes cellular proliferation in Burkitt lymphoma (BL), but also induces cell cycle arrest and apoptosis mediated by TP53, a tumor suppressor gene that is mutated in 40% of BL cases. To identify therapeutic targets in BL, we investigated molecular dependencies in BL cell lines using RNAi-based, loss-of-function screening. By integrating genotypic and RNAi data, we identified a number of genotype-specific dependencies including the dependence of TCF3/ID3 mutant cell lines on TCF3 and of MYD88 mutant cell lines on TLR signaling. TP53 wild-type (TP53wt) BL were dependent on MDM4, a negative regulator of TP53. In BL cell lines, MDM4 knockdown induced cell cycle arrest and decreased tumor growth in a xenograft model in a p53-dependent manner, while small molecule inhibition of the MDM4-p53 interaction restored p53 activity resulting in cell cycle arrest. Consistent with the pathogenic effect of MDM4 upregulation in BL, we found that TP53wt BL samples were enriched for gain of chromosome 1q which includes the MDM4 locus. 1q gain was also enriched across non-BL cancer cell lines (n=789) without TP53 mutation (23% in TP53wt and 12% in TP53mut, p<0.001). In a set of 216 cell lines representing 19 cancer entities from the Achilles project, MDM4 was the strongest genetic dependency in TP53wt cell lines (p<0.001).\n\nOur findings show that in TP53wt BL, MDM4-mediated inhibition of p53 is a mechanism to evade cell cycle arrest. The data highlight the critical role of p53 as a tumor suppressor in BL, and identifies MDM4 as a key functional target of 1q gain in a wide range of cancers, which is therapeutically targetable.

cancer biology↗