43 research outputs found
Alteration of Flavin Cofactor Homeostasis in Human Neuromuscular Pathologies
No full textThe aim of this short review chapter is to provide a brief summary of the relevance of riboflavin (Rf or vitamin B2) and its derived cofactors flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) for human neuromuscular bioenergetics.Therefore, as a completion of this book we would like to summarize what kind of human pathologies could derive from genetic disturbances of Rf transport, flavin cofactor synthesis and delivery to nascent apoflavoproteins, as well as by alteration of vitamin recycling during protein turnover
Alteration of Riboflavin transporter expression in human cancer
No full textRiboflavin (Rf), or vitamin B2, is an essential dietary component and represents the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important enzymatic cofactors required for terminal mitochondrial metabolism, and other cellular regulatory roles [1]. In different cells, Rf uptake occurs via specialized carrier-mediated processes performed by riboflavin transporter 1 (RFVT1; SLC52A1), RFVT2 (SLC52A2), and RFVT3 (SLC52A3) [2], belonging to the solute carrier family 52 (SLC52). Inside the cells, Rf is phosphorylated to FMN by riboflavin kinase (RFK, EC 2.7.1.26), and it is subsequently metabolized by FAD synthase (FADS, EC 2.7.7.2) to FAD, the flavin cofactor mainly located in mitochondria [1]. Rf pathway is gaining importance in the field of cancer therapy, since photoproducts are useful in preventing proliferation and metastasis of solid tumors [1]. An aberrant expression of RFVT3 is associated with stepwise development of esophageal squamous cell carcinoma (ESCC) [3]. Furthermore, a low intake of dietary Rf can lead to the development of colorectal cancer (CRC). Our group has demonstrated that alterations of transcription/translation of RFVTs make colon cancer cells greedy for the vitamin [4]. Here, we point our attention on the possible involvement of flavin cofactor homeostasis in other types of human cancer. To do this, we used both model cell lines and human biopsies. We correlated the altered expression of the three transporters with the increase of Rf-derived cofactor levels. The biochemical consequences of altering cellular flavoproteome will be discussed
Adapting cellular riboflavin transport and metabolism in pancreatic ductal adenocarcinoma
No full textRiboflavin (Rf) is an essential dietary component and is the precursor of FMN and FAD, the redox enzymatic cofactors required for mitochondrial terminal metabolism 1. Alterations in flavin homeostasis are associated with several pathological conditions, among which neuromuscular disorders 2 and cancer 3. We have recently demonstrated that transcription/translation of Rf transporters (RFVTs) are profoundly altered in colorectal cancer and we proposed that cancer cells need large amounts of Rf 4. Based on these evidences, we studied aspects related to flavin homeostasis in pancreatic ductal adenocarcinoma (PDAC) cells, comparing RFVT expression, the synthesis of FAD, and the amount of the mitochondrial flavoprotein, SDHA, in PANC-1 and in PANC-1 derived cancer stem cells (CSCs), which are expected to be metabolically plastic and malignant 5. As a control, we used HPDE (Human Pancreatic Ductal Epithelioid) cells which exhibit a gene expression pattern that more consistently resembles normal cell phenotype rather than cancerous ductal cells 6. The results obtained show that SDHA levels and flavin homeostasis are altered in tumour cells compared to HPDE, and that CSCs have higher rate of synthesis of flavin cofactors.
These data favour the hypothesis that the metabolic pathway responsible for FAD synthesis could represent a new therapeutic target to reduce the proliferation of CSCs
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
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
Exercise-induced rhabdomyolysis and transient loss of deambulation as outset of partial carnitine palmityl transferase II deficiency.
No full textWe report the case of a 13-year-old boy with an abrupt onset of leg pain and muscle weakness, incapability of deambulation and a laboratory picture of exercise-induced acute rhabdomyolysis. Intravenous hyperhydration and forced diuresis were adopted to avoid renal complications. No evidence of articular or residual muscular damage was appreciated in the short-term. The recurrence of rhabdomyolysis required a muscular biopsy showing a disturbance of fatty acid beta-oxidation pathway
Production of the recombinant human riboflavin transporters SLC52A1, 3 and functional assay in proteoliposomes
No full textRiboflavin, the FMN and FAD precursor, is a crucial vitamin in cell metabolism. Its adsorption and tissue distribution are mediated by tree membrane transporters namely RFVT1-3. Mutations of their genes are associated with Riboflavin Transporter Deficiency. Moreover, derangements of the level of these transporters have been found in several human cancers. To obtain a suitable experimental tool for studying the function of the single proteins, for testing the effect of pathological mutations and for validating predicted ligands as candidate drugs, we have set up a proteoliposome system harbouring the functional RFVT1 or RFVT3. RFVT proteins have been produced in E. coli and purified to the homogeneity by affinity chromatography. The purified proteins show an apparent molecular mass of 45.6 or 48.4 kDa, which are very close to the theoretical mass of RFVT1 or RFVT3, respectively. The purified transporters have been reconstituted into proteoliposomes using a methodology previously pointed out for RFVT2. The transport of riboflavin shows cooperative kinetics with K0.5 values of 0.86 or 1.13 μM and Hill coefficients of 1.19 or 1.3 for RFVT1 or RFVT3, respectively. The K0.5 data of both the transporters are similar the Km reported in intact cell studies. The transporters are inhibited by the riboflavin analogues FMN and lumiflavin in agreement with the molecular docking simulations
Efficacy of high-dose methylprednisolone pulses in a child with noninfectious persistent pleuropericarditis revealing systemic juvenile idiopathic arthritis
No full textA case of systemic-onset juvenile idiopathic arthritis is presented and successful treatment with methylprednisolone pulses is highlighted
FAD synthase deficiency: a severe mitochondrial myopathy involving a secondary reduction of RFVT2 expression
No full textThe redox cofactor FAD is essential for mitochondrial
functionality: in the inner- membrane it ensures the activity of the
respiratory chain complex II and of the ETF/ETFQO system, in the
matrix the oxidation of pyruvate and other α-oxoacids as well as of
some amino acids. Matrix located FAD-dependent dehydrogenases
are also involved in β-oxidation of fatty acil-CoAs.
The last step of the metabolic pathway converting the vitamin
riboflavin (Rf) into FAD is catalyzed by FAD synthase (FADS), coded
by human FLAD1 [1]. FLAD1 variations were identified as a cause of a
severe lipid storage myopathy resembling Multiple Acyl CoA
Dehydrogenase Deficiency, named LSMFLAD (OMIM #255100).
Patients’ symptoms can sometimes favourably respond to Rf therapy.
In the frame of the structural and functional characterisation of
the different hFADS isoforms, we describe here some morphological
and biochemical alterations in patients’ fibroblasts expressing FLAD1
pathological variants. In these cells we observed an impairment of
mitochondrial bioenergetics, mainly due to reduction in the level of
succinate dehydrogenase flavoprotein subunit, accompanied by, at
least in one patient, an increase in cellular ROS and a decrease of
mtDNA content. In the same patient’s cells, increased PGC-1α and
PrxIII levels were also observed, suggesting an active response to
stress conditions. Interestingly enough, in patient’s fibroblasts we
proved a drastic reduction of the levels of both the transcripts and
the protein product of SLC52A2 gene, i.e., RFVT2, the main Rf
transporter in muscle [1]. The decreased levels of all the flavin
species in the cell extracts are, thus, explainable as due to the
impairment of Rf flux from outside. In conclusion, a proposal is made
here that RFVT2 may be the primary target of Rf-based (and possibly
alternative) therapeutic strategies against LSMFLAD.
[1] M. Tolomeo, A. Nisco, P. Leone, M. Barile, Development of
Novel Experimental Models to Study Flavoproteome Alterations in
Human Neuromuscular Diseases: The Effect of Rf Therapy,
International journal of molecular sciences, 21 (2020)
Pursuing the Development of New Antiviral INSTIs
No full textThe Highly Active Antiretroviral Therapy (HAART) [1] consists of the combination of two or more drugs acting against three viral enzymes: reverse transcriptase, protease, and integrase (IN). The latter catalyzes integration of viral DNA with host DNA and was recently identified as a target for a promising class of drugs, Integrase Strand Transfer Inhibitors (INSTI).[2]
Currently, three drugs that seem to inhibit efficiently IN-HIV-1, namely Raltegravir, Elvitegravir and Dolutegravir, have been approved by FDA (American Food and Drug Administration) for therapeutic use.[3] Nevertheless, the outbreak of drug resistance during the therapeutic approach requires continuous and unceasing design of new antivirals.
Presently, our research efforts are focused on the development of new INSTI cytosine-based systems (as depicted in the figure) that originate from preliminary conformational analysis showing the core moiety to provide adequate facial orientation in the bioactive conformation, when functional groups suitable for insertion of additional structural elements are present in their molecules.
Under our conditions, a first generation of molecules have already been prepared and also tested to assess their biological activity in vitro, at Xpress Bio-laboratories (Maryland (USA).
Molecular docking analysis prompted the possibility to improve their potential inhibitory activity by structural modifications at the side chains and, therefore, we report herein the exploitation of our procedure to get a second generation of cytosine-based molecules that are presently under biological evaluation.
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