Nucleotide biosynthesis

Transcript

1 ucleotide biosynthesis Base ucleoside ucleotide (base + ribose) (nucleoside + phosphate) Adenine Adenosine AMP (adenylic acid or adenosine monophosphate) Guanine Guanosine GMP (guanylic acid or guanosine monophosphate) Uracil Uridine UMP (uridylic acid or uridine monophosphate) Thymine Thymidine TMP (thymidylic acid or thymidine monosphosphate) ytosine ytidine MP (cytidylic acid or cytidine monophosphate)

2 AD

3 rotico aciduria Malattia genetica rara caratterizzata da anemia per difetto di maturazione degli eritrociti e per la presenza di acido orotico nelle urine. I soggetti affetti sono incapaci di convertire l acido orotico in UMP Tipo 1: carenza dell enzima UMP sintasi (sia orotato fosforibosile transferasi che orotato monofosfato decarbossilasi) Tipo 2: difetto a carico solo dell orotato monofosfato decarbossilasi (terapia con uridina) Dosaggio acido orotico nelle urine PL 275 nm Più specifico L/MS/MS

4

5 Experiments with radioactive tracers showed that the purine ring is assembled from 5 different types of building block Amino group of aspartate 10 -formyl-tf 2 Glycine 10 -formyl-tf Amide of glutamine (TF = tetrahydrofolate) A common pathway of 11 steps beginning with phosphoribosylpyrophosphate (PRPP) and glutamine leads to inosine monophosphate (IMP). Separate branches then lead to AMP and GMP. PRPP + Gln IMP AMP GMP

6 1 PRPP sintasi 2 amidofosforibosile transferasi 3 GAR sintetasi 4 GAR transformilasi 5 FGAM sintasi 6 AIR sintasi 7 AIR carbossilasi 8 SAAIR sintetasi 9 Adenilsuccinatoliasi 10 AIAR transformilasi 11 IMP sintasi

7

8 Biosintesi dei deossiribonucleotidi e catabolismo delle purine

9 ADP, GDP, DP & UDP can be reduced to the corresponding deoxyribonucleotides ADP 2 dadp GDP dgdp DP ddp UDP X S S ribonucleotide reductase X S S dudp ucleoside diphosphates are reduced by cysteines in the enzyme, ribonucleotide reductase. ADP + ADP Regeneration of the reduced enzyme requires several steps) dttp DA replication also requires thymine, which is made from dudp.

10 Primates oxidize purines to uric acid for excretion adenosine ribose guanosine ribose Birds, reptiles and insects also excrete uric acid. Fish and most terrestrial mammals oxidize uric acid further before excreting the product. uric acid uric acid has several tautomeric forms

11 Uric acid production from purines adenosine inosine hypoxanthine 2 ribose adenosine deaminase ribose P i ribose-1-p 2 - guanosine ribose xanthine xanthine oxidase uric acid Xanthine oxidase contains FAD, a Mo complex, and two Fe-S centers. Its substrates are the free purines, not nucleosides or nucleotides. xanthine oxidase 2 2

12 deoxyadenosine Mutations in adenosine deaminase cause Severe ombined Immunodeficiency Disease SIDS adenosine deaminase uric acid deoxyribose deoxyribose datp In the absence of adenosine deaminase, datp accumulates to high levels. The datp inhibits ribonucleotide reductase, preventing synthesis of the other deoxynucleotides and cutting off DA synthesis. Lymphocytes are particularly susceptible to this inhibition. Untreated SID is fatal. SID was one of the first disorders to be addressed by gene therapy. ribonucleotides - ribonucleotide reductase deoxyribonucleotides DA

13 Deposition of sodium urate crystals in tissues causes gout Impaired excretion of uric acid results in high levels of uric acid in body fluids. rystals of sodium urate form in the toe, kidney and other tissues, causing painful inflamation. hypoxanthine xanthine uric acid xanthine oxidase xanthine oxidase allopurinol Gout can be treated with allopurinol, an analog of hypoxanthine that inhibits xanthine oxidase. sodium urate a + -

14 Gotta: dal latino «gutta» (= goccia) Già descritta da Ippocrate (ca. 460 ca. 370 a..), Galeno ( d..) e Paracelso ( ) Definizione: Artropatia cristallina con deposito intra- ed extrarticolare di cristalli di acido urico, in presenza di un difetto del metabolismo delle purine Deposito di cristalli di acido urico: elle articolazioni «vera» artropatia urica ei tessuti possibile formazione di tofi (noduli) Spesso associata ad una nefropatia urica, disturbi del metabolismo del glucosio e/o dei grassi

15 Diagnostica Laboratorio Puntato: tipici cristalli di acido urico, evidenziati al microscopio con luce polarizzata (rifrazione neg.) Escissione di tofi delle parti molli: valutazione al microscopio del materiale biancastro estratto e dimostrazione di cristalli di acido urico in massa Sangue: nell attacco acuto di gotta nel 90% dei casi vi è un iperuricemia. Inoltre aumento dei parametri fiammatori (VES e PR, leucocitosi)

16

17

18 Esami necessari per chiarire la causa di un iperuricemia Acido urico sierico reatinina sierica Eliminazione dell acido urico e della creatinina nelle urine delle 24 h (clearance)

19 Salvage pathways capture free purine and pyrimidine bases and regenerate nucleotides adenine 2 adenine phosphoribosyl transferase P PP i P - 2 P P - AMP 5-phosphoribosyl-1- pyrophosphate (PRPP) Another enzyme (hypoxanthine-guanine phosphoribosyl transferase) works on hypoxanthine and guanine. Mutations in this enzyme lead to Lesch-yhan syndrome.

20 Lesch-yhan syndrome Lesch-hyan syndrome, an X-linked disorder that results from a mutation in the gene for hypoxanthine-guanine ribosylphosphotransferase, is characterized by severe neurological defects. mental retardation self-mutilation cerebral palsy elevated uric acid (gout)

21 Lesch-yhan Syndrome can be diagnosed prenatally Fetal fibroblasts obtained by amniocentesis are cultured in the presence of 3 -hypoxanthine. ells from normal individuals convert hypoxanthine into IMP, which procedes to AMP and GMP and is incorporated into DA. normal hypoxanthine PRPP PP i P - 2 IMP Lesch-yhan Blocked in Lesch-yhan syndrome DA AMP GMP