883 research outputs found
Ferritin engineering by chemical modification for bioimaging and drug delivery
No full textNegli ultimi anni, le nanoparticelle di ferritina hanno ricevuto crescente attenzione per le loro peculiari proprietà ed il loro impiego per applicazioni diagnostiche e terapeutiche. Le ferritine infatti sono nanoparticelle proteiche stabili, biocompatibili, versatili, uniformi ed omogenee con 24 subunità assemblate in modo da formare una gabbia sferica, con una cavità interna di 8 nm di diametro, in grado di ospitare qualsivoglia molecola. Inoltre le ferritine possono essere internalizzate all’interno delle cellule mediante endocitosi mediata dal recettore TfR1, un recettore largamente sovraespresso nelle cellule cancerose. Per queste caratteristiche le ferritine emergono come nanoparticelle ideali per l’incapsulamento ed il delivery selettivo di varie molecole endogene. Tuttavia le ferritine di mammifero sono assemblate in una forma stabile e chiusa di tetraeicosamero, in grado di dissociare solo in condizioni estreme quali pH acidi potenzialmente dannosi sia per la proteina che per le molecole incapsulate. Al contrario, la ferritina di Archaeaoglobus fulgidus (AfFt) è in grado di dissociare e riassociare dipendentemente dalla forza ionica dell’ambiente in cui si trova. Tale ferritina è stata perciò “umanizzata” mediante tecniche di ingegneria genetica producendo un mutante di superficie di AfFt con un loop esterno in grado di mimare il motivo di riconoscimento della ferritina umana per il recettore TfR1 in cellule umane e con l’architettura e le proprietà di oligomerizzazione tipiche della ferritina di AfFt. Le proprietà di associazione e dissociazione dipendenti dalla forza ionica del sistema sono state attentamente caratterizzate da un punto di vista sia termodinamico che cinetico, tramite funzionalizzazione della proteina con molecole fluorescenti di pirene. Tale fluoroforo è inoltre stato utilizzato per la visualizzazione, mediante la tecnica di microscopia di fluorescenza a due fotoni, dell’uptake di ferritina all’interno di cellule cancerose quali le cellule HeLa. L’effettiva internalizzazione della ferritina dimostra quindi sia l’avvenuto riconosciuto del mutante umanizzato da parte del recettore TfR1 sia il potenziale di tale nanoparticella per applicazioni di microscopia e bioimaging. In aggiunta l’utilizzo della ferritina umanizzata come nano particella per l’incapsulamento ed il delivery selettivo su cellule cancerose di brevi sequenze di DNA è sotto sperimentazione.
In conclusione questi studi dimostrano la potenzialità della ferritina umanizzata come nano particella versatile, facilmente modificabile ed in grado di incapsulare molecole all’interno della cavità in condizioni fisiologiche al fine di trasportare e rilasciare selettivamente la molecola incapsulata all’interno delle cellule tumorali per scopi terapeutici o diagnostici
Supplemental_Material – Supplemental material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics
No full textSupplemental material, Supplemental_Material for OCTN: A Small Transporter Subfamily with Great Relevance to Human Pathophysiology, Drug Discovery, and Diagnostics by Lorena Pochini, Michele Galluccio, Mariafrancesca Scalise, Lara Console and Cesare Indiveri in SLAS Discovery</p
Functional Study of the Human Riboflavin Transporter 2 Using Proteoliposomes System
No full textRiboflavin is essential for cell viability. The biologically active forms of riboflavin, FMN and FAD, participate in many biochemical redox reactions including the metabolism of carbohydrates, amino acids, and lipids. Differently from bacteria, fungi, and plants which synthesize riboflavin, higher organisms have lost the ability to synthesize the vitamin and must absorb it from food and intestinal microflora production. The riboflavin flux through cell membranes occurs via specific transporters belonging to the SLC52 family. Three members of this family have been identified so far which show poor homology with the riboflavin transporters of Saccharomyces cerevisiae or bacteria. Alterations of RFVTs are causative of severe diseases. Indeed, under pathological stress, humans are susceptible of developing riboflavin deficiency. Such a deficiency in pregnancy induces fetus abnormalities, and has been indicated as a risk factor for anemia, cancer, cardiovascular diseases, and neurodegeneration. Moreover, inherited diseases are also of interest; the most well-described is the Brown-Vialetto-van Laere syndrome, a rare neurological disorder characterized by infancy onset sensorineural deafness and pontobulbar palsy. Numerous polymorphisms of Slc52a2 and Slc52a3 genes associated with this syndrome have been discovered. In spite of their important metabolic role and their relevance to human health, the riboflavin transporters are still poorly characterized. Bacterial overexpression, purification, and protein reconstitution in liposomes represent an up-to-date methodology for obtaining functional data information. The methodology for reconstituting the RFVT2 into proteoliposomes and performing transport assay is described. These methods will be suitable for investigating the functional defects of the variants of RFVTs associated with human pathologies
A novel spiking CPG-based implementation system to control a lamprey robot
No full textThe study proposed describes preliminary results of a spiking implementation of lamprey's Central Pattern Generator (CPG) using Neuromorphic VLSI devices. Several robotic lamprey implementations have been built to test the models in a bio-mimetic artifact but, in these systems there is a clear separation between the mechanical system, and their control part. This study aims to implement a CPG hardware network, to directly control actuators, creating a biomimetic robot both from mechanical and electronic point of view
FAD forming and destroying via human FAD synthase: a puzzle of modulated and modulating activities
No full textFAD synthase (FADS or FMN:ATP adenylyl transferase) coded by human FLAD1 gene, is the last enzyme in the pathway converting riboflavin into the redox cofactor FAD.Mutations in FLAD1 gene are responsible for Riboflavin-Responsive and Non-Responsive MADD and Combined Respiratory-Chain Deficiency[1].
Alternative splicing of the FLAD1 gene generates different hFADS isoforms: the mitochondrial isoform 1 and the cytosolic isoform 2. These are bi-functional enzymes containing two domains: a PAPS domain at the C-terminus able to catalyze FAD synthesis (EC 2.7.7.2), a molybdopterin-binding domain at the N-terminus able to perform FAD hydrolysis (EC 3.6.1.18).We show here that theCo2+-dependent hydrolytic activity of hFADS2 is strongly stimulated in the presence of K+, reaching a Vmax even higher than that of FAD synthesis. hFADS2acts as a non-NuDiX hydrolaseand it could interconnect FAD and NAD homeostasis.
Recently,in patients suffering for frameshift mutations in the FLAD1 gene, we revealed ashort transcript variant corresponding to isoform 6,containing the sole PAPS domain[1]. We overproduced and characterized this emergency protein, which is relevant for patient survival. It is able to synthesize, but not to hydrolyze FAD [2].In the aim to find a target for therapy intervention in patients harboring FADS defects, a variant of hFADS6 carrying the site-directed mutation D238A, expected to exhibit a higher Kcat, was also over-produced, purified and characterized
MODULATING CELLULAR FLAVIN COFACTOR LEVELS IN HUMANS VIA THE BI-FUNCTIONAL AND MONOFUNCTIONAL FLAD1 GENE PRODUCTS
No full textA spiking implementation of the lamprey's Central Pattern Generator in neuromorphic VLSI
No full textDISC867317_SupplementalMaterial – Supplemental material for SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure
No full textSupplemental material, DISC867317_SupplementalMaterial for SLC6A14, a Pivotal Actor on Cancer Stage: When Function Meets Structure by Luca Palazzolo, Chiara Paravicini, Tommaso Laurenzi, Sara Adobati, Simona Saporiti, Uliano Guerrini, Elisabetta Gianazza, Cesare Indiveri, Catriona M. H. Anderson, David T. Thwaites and Ivano Eberini in SLAS Discovery</p
Human riboflavin transporters in health and diseases
No full textRiboflavin, otherwise known as vitamin B2, is an essential dietary component and it represents the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), the redox enzymatic cofactors required for hundreds of enzymes involved in protein folding, apoptosis, epigenetics and mitochondrial terminal metabolism [1]. Flavin cofactor biosynthesis in different cells starts from riboflavin uptake, which occurs via specialized carrier-mediated processes which are supported by three specific members of the solute carrier family 52 (SLC52A), identified and named respectively RFVT1, RFVT2 and RFVT3. We pointed our attention on a profound alteration of flavin cofactor homeostasis in human colorectal [2] and some other types of cancer, which are accompanied by dysregulation of RFVTs expression. Changing the level of RFVTs expression as a possible mean to reprogram cellular flavoproteome will be discussed. Primary or secondary alterations of the activity/expression of RFVTs have been correlated with rare inherited neuro-muscular disorders, some of which treatable with high doses of the vitamin [3]. The effect of alteration of flavin cofactor availability on mitochondrial flavoenzymes in human cells and worm models will be discussed. To better study structure-function relationships in these human diseases over-production of the human RFVT2 transporter in E. coli was carried out and reconstitution of the purified protein in proteoliposomes for transport assay was performed
The Expression of Riboflavin Transporters in Human Cancer
No full textRiboflavin (Rf), otherwise known as vitamin B2, is an essential dietary component and represents
the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), that are
important enzymatic cofactors required for carbohydrate, amino acid and lipid metabolism, and
other cellular regulatory roles. Humans, being unable to synthesize riboflavin, must obtain it from
food, in particular from milk, meats, fatty fish and green vegetables, and, to a lesser amount, from
intestinal microflora. After intestinal absorption, Rf is distributed from the blood to several tissues:
Rf cellular uptake occurs via specialized carrier-mediated processes supported by three specific
members of the solute carrier family 52 (SLC52A), identified and named Rf transporter 1 (RFVT1;
SLC52A1), 2(RFVT2;SLC52A2), and 3(RFVT3; SLC52A3), respectively [1, 2]. Once inside the cells, Rf is
phosphorylated to FMN by riboflavin kinase (E.C. 2.7.1.26) and it is subsequently metabolized to
FAD by FAD synthase (E.C. 2.7.7.2), existing in different isoforms localised in cytosol, mitochondrion
and nucleus [3].
A low intake of dietary Rf can lead to negative health consequences, that include the development
of colorectal cancer (CRC). We have recently demonstrated that alterations of
transcription/translation of RFVTs, lead colon cancer cells to become greedy for the vitamin [4].
The aim of the present study was to investigate whether the expression levels of RFVTs are also
altered in uterus and breast cancers. To do this, we used both cell models and human biopsies.
We correlated altered expression of translocators with the increase of Rf-derived cofactor levels.
The biochemical consequences of altering cellular flavoproteome will be discussed
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