Adenine Phosphoribosyltransferase Research Articles

Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

The Chinese cabbage is a crop belonging to the family Cruciferae; very few studies have focused on the analysis of gene expression in this economically important crop. In this study, we used real-time quantitative polymerase chain reaction to identify the control genes that are the most stably expressed in a given set of tissues and under conditions of drought stress and downy mildew infection. We characterized the transcript stability of nine candidate reference genes, namely, those encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (Ubc), elongation-factor-1-α (EF-1-α), adenine phosphoribosyltransferase (Apr), clathrin, cyclophilin (Cyp), tubulin (Tub), Actin, and 18s rRNA, using the geNorm and Normfinder software programs. The results of a geNorm analysis indicated that EF-1-α and Apr were the most suitable reference genes among the given set of tissues and that GAPDH and Ubc were the most stable genes under conditions of drought stress and downy mildew infection. Furthermore, the results of an analysis performed using the Normfinder software program indicated EF-1-α to be the best normalization factor in the given set of tissues and under conditions of downy mildew infection and GAPDH to be the best factor under drought stress conditions. The results of the geNorm analysis also helped determine the minimum number of genes required to calculate a reliable normalization factor. A study on the variability of expression of PSY expression showed that the relative quantification of this gene varied depending on the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

Urinary stones resembling uric acid stones.

A 52-year-old man was referred for the evaluation of mild renal failure with severe hypertension (220/130mmHg), which was resistant to combination therapy of a calcium channel blocker and an angiotensin II receptor blocker. Although he reported no past history indicating urolithiasis, urinalysis revealed plenty of round brownish stones (Figure 1A). The stones resembled uric acid (UA) stones (Figure 1B), but most of them had spicules radiating from the centre and rough surface, which are not prominent in UA stones. Furthermore, the stones were friable compared with UA stones. From these characteristics, the stones were recognized as 2,8-dihydroxyadenine (2,8-DHA) stones [1,2]. The diagnosis of type II adenine phosphoribosyltransferase (APRT) deficiency was confirmed by the genomic analysis that revealed APRT*J homozygote (Figure 2) [3]. The treatment by allopurinol with a low purine and low sodium diet diminished the stones and reduced the systemic blood pressure to 140/80mmHg. In APRT deficiency, the oxidation to 2,8-DHA is the met-

Phenotype and Genotype Characterization of Adenine Phosphoribosyltransferase Deficiency

Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder causing 2,8-dihydroxyadenine stones and renal failure secondary to intratubular crystalline precipitation. Little is known regarding the clinical presentation of APRT deficiency, especially in the white population. We retrospectively reviewed all 53 cases of APRT deficiency (from 43 families) identified at a single institution between 1978 and 2009. The median age at diagnosis was 36.3 years (range 0.5 to 78.0 years). In many patients, a several-year delay separated the onset of symptoms and diagnosis. Of the 40 patients from 33 families with full clinical data available, 14 (35%) had decreased renal function at diagnosis. Diagnosis occurred in six (15%) patients after reaching ESRD, with five diagnoses made at the time of disease recurrence in a renal allograft. Eight (20%) patients reached ESRD during a median follow-up of 74 months. Thirty-one families underwent APRT sequencing, which identified 54 (87%) mutant alleles on the 62 chromosomes analyzed. We identified 18 distinct mutations. A single T insertion in a splice donor site in intron 4 (IVS4 + 2insT), which produces a truncated protein, accounted for 40.3% of the mutations. We detected the IVS4 + 2insT mutation in two (0.98%) of 204 chromosomes of healthy newborns. This report, which is the largest published series of APRT deficiency to date, highlights the underdiagnosis and potential severity of this disease. Early diagnosis is crucial for initiation of effective treatment with allopurinol and for prevention of renal complications.

Two families with compound heterozygosity for adenine phosphoribosyltransferase deficiency

Adenine phosphoribosyltransferase deficiency is a disorder in which 2,8-dihydroxyadenine (2,8-DHA) crystalluria is caused by a congenital deficiency in the enzyme adenine phosphoribosyltransferase (APRT). In most cases, APRT deficiency is caused by autosomal recessive inheritance of a homozygote of the mutant gene APRT*Q0 or APRT*J, but there are also some cases in which the disorder is caused by the compound heterozygote APRT*Q0 and APRT*J. In the patients described here, brown round crystals were found in their urinary sediment. Crystalluria was the first sign of APRT deficiency, thereafter confirmed by genetic screening for APRT*/Q0 and APRT*. We performed genetic screening for APRT*Q0 and APRT*J in two families and diagnosed three cases of APRT*Q0 /APRT*J compound heterozygote-type APRT deficiency. Genetic screening for APRT*Q0 and APRT*J of family members is effective for early diagnosis and early treatment for family members.

Adenine metabolism in Plasmodium falciparum

Plasmodium falciparum lacks the de novo purine biosynthesis pathway and relies entirely on the salvage pathway to meet its purine nucleotide requirements. The entire flux for purine nucleotide biosynthesis in the parasite is believed to be through hypoxanthine guanine phosphoribosyltransferase (HGPRT), with the enzymes, adenosine kinase and adenine phosphoribosyltransferase (APRT) being unannotated in the Plasmodium genome database. This manuscript reports on the studies carried out to explore bypass mechanisms, if any, for AMP synthesis in the intraerythrocyitc stages of the parasite life cycle. Uptake and subsequent incorporation of radiolabel adenine in the nucleotide pool of saponin released erythrocyte free parasites implicated the role of parasite encoded enzymes in adenine metabolism. To explore the route for AMP synthesis in the parasite, we have monitored adenine mediated supplementation of metabolic viability in saponin released hadacidin ( N-formyl- N-hydroxyglycine) treated parasites. Our results implicate the role of an APRT like activity that enables parasite survival when the flux through the HGPRT pathway is blocked.

Crystalline nephropathy due to 2,8-dihydroxyadeninuria: an under-recognized cause of irreversible renal failure

2,8-dihydroxyadeninuria (DHA) disease (also called 2,8 dihydroxyadeninuria) is a rare autosomal recessive disorder caused by complete adenine phosphoribosyltransferase deficiency and typically manifests as recurrent nephrolithiasis. Only rare cases of DHA nephrolithiasis have been reported from the USA. Herein, we report three American patients who developed DHA crystalline nephropathy leading to end-stage renal disease (ESRD) with recurrence in the allograft. Three cases of DHA crystalline nephropathy were identified from the Renal Pathology Laboratory of Mayo Clinic. Detailed clinical and pathologic descriptions are provided. All three patients were Caucasian adults with no history of obstructive nephropathy. Two patients had no history of nephrolithiasis and one had a single episode of stones 36 years prior to presentation. All patients presented with severe renal failure with a mean serum creatinine of 7.5 mg/dl. Renal biopsies revealed numerous tubular and interstitial brown DHA crystals, tubular degenerative changes and moderate to marked tubulointerstitial scarring. Two patients were initially misdiagnosed, one as primary hyperoxaluria and the other as chronic interstitial nephritis. All three patients progressed to ESRD, within 1 month following renal biopsy in two and after 9 months in one. All three patients underwent renal transplantation with early disease recurrence in three allografts in two patients. DHA disease is an under-recognized condition that can lead to irreversible renal failure and frequently recurs in the transplant. It should be included in the differential diagnosis of crystalline nephropathy, even in the absence of history of nephrolithiasis.

2,8-Dihydroxyadenine Nephrolithiasis Induces Developmental Stage-specific Alterations in Gene Expression in Mouse Kidney

To identify factors that may be crucial for the initiation and progression of stone-induced injury in the developing mouse kidney by a prospective observational study using microarray analysis. Kidney stone diseases are common in premature infants, but the underlying molecular and cellular mechanisms are not fully defined. Mice with adenine phosphoribosyltransferase deficiency develop 2,8-dihydroxyadenine (DHA) nephrolithiasis. The gene expression changes between Aprt(-/-) and Aprt(+/+) kidneys from newborn and adult mice were compared using Affymetrix gene chips. Targets of interest were further analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. We identified a set of genes that were differentially expressed in the developing kidney in response to DHA-induced injury. In 1-week-old Aprt(-/-) mice, the expression of Sprr2f and Clu was highly augmented and that of Egf was significantly decreased. We also observed that maturation-related gene expression changes were delayed in developing Aprt(-/-) kidneys, and immature Aprt(-/-) kidneys contained large numbers of intercalated cells that were blocked from terminal differentiation. This study presents a comprehensive picture of the transcriptional changes induced by DHA stone injury in the developing mouse kidney. Our findings help explain growth impairment in kidneys subject to injury during the early stages of development.

Selective Killing of Tumors Deficient in Methylthioadenosine Phosphorylase: A Novel Strategy

BackgroundThe gene for methylthioadenosine phosphorylase (MTAP) lies on 9p21, close to the gene CDKN2A that encodes the tumor suppressor proteins p16 and p14ARF. MTAP and CDKN2A are homozygously co-deleted, with a frequency of 35 to 70%, in lung and pancreatic cancer, glioblastoma, osteosarcoma, soft-tissue sarcoma, mesothelioma, and T-cell acute lymphoblastic leukemia. In normal cells, but not in tumor cells lacking MTAP, MTAP cleaves the natural substrate, 5′-deoxy-5′-methylthioadenosine (MTA), to adenine and 5-methylthioribose-1-phosphate (MTR-1-P), which are then converted to adenine nucleotides and methionine. This distinct difference between normal MTAP-positive cells and tumor MTAP-negative cells led to several proposals for therapy. We offer a novel strategy in which both MTA and a toxic adenine analog, such as 2,6-diaminopurine (DAP), 6-methylpurine (MeP), or 2-fluoroadenine (F-Ade), are administered. In MTAP-positive cells, abundant adenine, generated from supplied MTA, competitively blocks the conversion of an analog, by adenine phosphoribosyltransferase (APRT), to its active nucleotide form. In MTAP-negative tumor cells, the supplied MTA cannot generate adenine; hence conversion of the analog is not blocked.Principal FindingsWe show that this combination treatment – adenine analog plus MTA – kills MTAP-negative A549 lung tumor cells, while MTAP-positive human fibroblasts (HF) are protected. In co-cultures of the breast tumor cell line, MCF-7, and HF cells, MCF-7 is inhibited or killed, while HF cells proliferate robustly. 5-fluorouracil (5-FU) and 6-thioguanine (6-TG) may also be used with our strategy. Though neither analog is activated by APRT, in MTAP-positive cells, adenine produced from supplied MTA blocks conversion of 5-FU and 6-TG to their toxic nucleotide forms by competing for 5-phosphoribosyl-1-pyrophosphate (PRPP). The combination of MTA with 5-FU or 6-TG, in the treatment of MTAP-negative tumors, may produce a significantly improved therapeutic index.ConclusionWe describe a selective strategy to kill tumor cells lacking MTAP.

Adenine Phosphoribosyltransferase Deficiency in a Chinese Man with Early-onset Gout

To the Editor: Early-onset gout is known to be often related to genetic factors. However, the molecular genetic issues are not understood for these patients. In 2007, a 25-year-old Hong Kong Chinese man was referred for advice on his early-onset gout. The first of his many attacks was at age 18 years. A severe attack appeared at his right first metatarsophalangeal joint when he was 22 years old. His serum uric acid then was 600 μmol/l (10 mg/dl) and serum creatinine was 1.1 mg/l. His creatinine clearance was about 80 cc/min 3 years ago compared to 88 cc/min 10 years ago. He had 24-h uric acid excretion (using a low purine diet) at 5625 μmol/day (940 mg/day), indicating overproduction of uric acid. The treatment of choice for gout with overproduction of uric acid is allopurinol, but the patient was worried about the safety of allopurinol because of his allergic history to other drugs and his father’s allergy to allopurinol. We suggested that the test of HLA-B*5801 might be … Address reprint requests to Dr. Chen E-mail: chungjen{at}adm.cgmh.org.tw

Polypurine Hairpins Directed against the Template Strand of DNA Knock Down the Expression of Mammalian Genes

We analyzed whether polypurine hairpins (PPRHs) had the ability to knock down gene expression. These hairpins are formed by two antiparallel purine domains linked by a loop that allows the formation of Hoogsteen bonds between both domains and Watson-Crick bonds with the target polypyrimidine sequence, forming triplex structures. To set up the experimental conditions, the human dhfr gene was used as a model. The PPRHs were designed toward the template strand of DNA. The transfection of the human breast cancer cell line SKBR3 with these template hairpins against the dhfr gene produced higher than 90% of cell mortality. Template PPRHs produced a decrease in DHFR mRNA, protein, and its corresponding enzymatic activity. In addition, the activity of DHFR PPRHs was tested against breast cancer cells resistant to methotrexate, observing high cell mortality. Given the difficulty in finding long polypyrimidine stretches, we studied how to compensate for the presence of purine interruptions in the polypyrimidine target sequence. The stability of PPRH was measured, resulting in a surprisingly long half-life of about 5 days. Finally, to test the generality of usage, template PPRHs were employed against two important genes involved in cell proliferation, telomerase and survivin, producing 80 and 95% of cell death, respectively. Taken together our results show the ability of antiparallel purine hairpins to bind the template strand of double strand DNA and to decrease gene transcription. Thus, PPRHs can be considered as a new type of molecules to modulate gene expression.

Fertility Alternation of Thermo-Photo-Sensitive Genic Male Sterile (TGMS) Wheat Line C412S and its Association with Adenine Phosphoribosyltransferase Gene Expression

C412S, a novel thermo-photo-sensitive genic male sterile (TGMS) wheat ( Triticum aestivum L.) line, is derived from a cross between TGMS line C49S and the conventional line C412. The objectives of this study were to examine the fertility alternation of C412S planted at different sowing dates and to investigate the possible function of APRT1 and APRT2 in the fertility alternation of thermo-photo-sensitive wheat. Under the 10 treatments of seeding date from 26 October to 10 December in 2005–2007 growing seasons, C412S showed fertility variations from complete sterility to high sterility, semisterility, low sterility, and normal fertility, which were achieved by adjusting the environmental temperature and photoperiod conditions during the development of male sex cells. Smear and squash microscopic technique was employed to follow the developmental progress of pollen mother cell (PMC) meiosis and microsporogenesis. Fertility-sensitive period of C412S was from PMC formation stage to mature pollen stage, especially from PMC formation to PMC meiosis stage. C412S pollen abortion, which belongs to spherical abortion type, reached peak level at later mononucleate stage of microsporogenesis. Moreover, the full length of adenine phosphoribosyltransferase gene ( APRT) was cloned from C412S and its control line C412. Sequences analysis showed that there were a few base differences in APRT1 between C412S and C412. However, the deducted amino acid sequences of C412S and C412 are identical. According to the results of semiquantitative reverse transcription PCR (RT-PCR), the transcriptional level of APRT genes in the young spikes of the sterile line C412S was lower than that of the fertile line C412 from PMC formation to mononucleate microspore stage. This indicates that the transcriptional level of APRT in young spikes may associate with fertility alternation in TGMS wheat.

Genetic control of metabolism of mutagenic purine base analogs 6-hydroxylaminopurine and 2-amino-6-hydroxylaminopurine in yeast Saccharomyces cerevisiae

The influence of inactivation of genes, which control biosynthesis of inosine monophosphate (IMP) de novo and the purine utilization and interconversion pathway, on sensitivity of yeast Saccharomyces cerevisiae cells to the mutagenic and toxic action of 6-hydroxylaminopurine (HAP) and 2-amino-6-hydroxylaminopurine (AHA) was studied. It was shown that the manifestation of HAP and AHA mutagenic properties involves the action of enzyme adenine phosphoribosyltransferase encoded in yeast by APT1 gene. A blockade of each stage of IMP biosynthesis, with the exception of the block mediated by inactivation of genes ADE16 and ADE17 leading to the accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), was shown to enhance yeast cell sensitivity to the HAP mutagenic effect; however, it does not affect the sensitivity to AHA. A blockade of conversion of IMP into adenosine monophosphate (AMP) causes hypersensitivity of yeast cells to the mutagenic action of HAP and to the toxic effect of HAP, AHA, and hypoxanthine. It is fully probable that this enhancement of sensitivity to HAP and AHA is conditioned by changes in the pool of purines. This indicates that genes ADE12, ADE13, AAH1, and HAM1 controlling processes of purine utilization and interconversion in yeast make the greatest contribution to the system of protection against the toxic and mutagenic action of the examined analogs. Possible mechanisms of HAP detoxication in bacteria, yeast, and humans are considered.

Detecting Ricin: Sensitive Luminescent Assay for Ricin A-Chain Ribosome Depurination Kinetics

Ricin is a family member of the lethal ribosome-inactivating proteins (RIP) found in plants. Ricin toxin A-chain (RTA) from castor beans catalyzes the hydrolytic depurination of a single base from a GAGA tetraloop of eukaryotic rRNA to release a single adenine from the sarcin-ricin loop (SRL). Protein synthesis is inhibited by loss of the elongation factor binding site resulting in cell death. We report a sensitive coupled assay for the measurement of adenine released from ribosomes or small stem-loop RNAs by RTA catalysis. Adenine phosphoribosyl transferase (APRTase) and pyruvate orthophosphate dikinase (PPDK) convert adenine to ATP for quantitation by firefly luciferase. The resulting AMP is cycled to ATP to give sustained luminescence proportional to adenine concentration. Subpicomole adenine quantitation permits the action of RTA on eukaryotic ribosomes to be followed in continuous, high-throughput assays. Facile analysis of RIP catalytic activity will have applications in plant toxin detection, inhibitor screens, mechanistic analysis of depurinating agents on oligonucleotides and intact ribosomes, and in cancer immunochemotherapy. Kinetic analysis of the catalytic action of RTA on rabbit reticulocyte 80S ribosomes establishes a catalytic efficiency of 2.6 x 10(8) M(-1) s(-1), a diffusion limited reaction indicating catalytic perfection even with large reactants.

A 57-gene expression signature in B-cell chronic lymphocytic leukemia

B-CLL is the most frequent type of leukemia in the Western countries. The disease, common among the elderly, follows a variable course in terms of survival time and symptoms. There is evidence that the accumulation of lymphocytes in peripheral blood and bone marrow is due to a cell resistance to apoptosis rather than to highly proliferative cells. Genetic mechanisms that lead to the development and progression of disease are mainly unknown, although a number of prognostically and diagnostically important genetic markers have been identified. The aim of this study is to investigate the gene expression profile, by a specific chip for microarray analysis, in B-CLL lymphocytes with regard to factors involved in apoptosis cascade, signal transduction, purine metabolism enzymes, interleukin expression, enzymes involved in the responses to oxidative stress. We found relevant results in a set of 19 of the 57 genes considered. IMP dehydrogenase, adenine phosphoribosyltransferase, adenylosuccinate lyase, adenylate kinase, ADORA1, G-protein-coupled receptor kinase 6, Bcl-2-like 1 isoform 2, caspase 6, and 8 were found underexpressed; while ADORA3, Gars-Airs-Gart, adenylate kinase 3, adenylate deaminase, NMN adenylyltransferase, CD26, CD38, interleukins 18 and 4 were found overexpressed. The microarray technique is a powerful method for identification of potential important diagnostic and prognostic markers, besides giving prominence to genes candidate for further studies.

Progressive renal dysfunction and macrophage infiltration in interstitial fibrosis in an adenine-induced tubulointerstitial nephritis mouse model

Adenine phosphoribosyltransferase deficiency in mice or an excessive oral intake of adenine leads to the accumulation of 2,8-dihydroxyadenine (DHA) in renal tubules and that causes progressive renal dysfunction accompanied by interstitial fibrosis. However, the precise mechanism responsible for DHA-induced progressive fibrosis is not fully understood. The present study investigates the possible involvement of monocytes/macrophages in the progressive fibrosis induced by feeding adenine to mice. Urinary calculi were deposited in tubules on day 7 after the initiation of adenine feeding. Elevation of the serum creatinine level and loss of body weight were observed in a time-dependent manner, suggesting the development of typical renal dysfunction induced by the adenine feeding. In renal tissue, mRNA expression of MCP-1, MIP-1alpha, RANTES, IL-1beta, CCR2, TGF-beta, alpha-smooth muscle actin (alpha-SMA) and collagen 1a1 was increased in parallel. Along with the increased expression of these genes, a remarkable infiltration of macrophages into the tubulointerstitial area was observed in a time-dependent manner. In addition, in the tubulointerstitial area, alpha-SMA positive fibroblasts were increased in parallel with collagen deposition. These results suggest that the excessive consumption of adenine leads to progressive renal dysfunction in mice. We speculate that the accumulation of DHA in tubules might stimulate epithelium to produce MCP-1 and that profibrogenic TGF-beta produced by infiltrated macrophages might stimulate interstitial fibroblasts to produce collagen. These results indicate that macrophage infiltration is one of the triggers that initiates interstitial fibroblast activation and collagen deposition followed by renal dysfunction.

Nephrolithiasis related to inborn metabolic diseases

Nephrolithiasis associated with inborn metabolic diseases is a very rare condition with some common characteristics: early onset of symptoms, family history, associated tubular impairment, bilateral, multiple and recurrent stones, and association with nephrocalcinosis. The prognosis of such diseases may lead to life threatening conditions, not only because of unabated kidney damage but also because of progressive extra-renal involvement, either in a systemic form (e.g. primary hyperoxaluria type 1, requiring combined liver and kidney transplantation), or in a neurological form (Lesch–Nyhan syndrome leading to auto-mutilation and disability, phosphoribosyl pyrophosphate synthetase superactivity, which is associated with mental retardation). Patients with other inborn metabolic diseases present only with recurrent stone formation, such as cystinuria, adenine phosphoribosyl-transferase deficiency, xanthine deficiency. Finally, nephrolithiasis may be secondarily part of some other metabolic diseases, such as glycogen storage disease type 1 or inborn errors of metabolism leading to Fanconi syndrome (nephropathic cystinosis, tyrosinaemia type 1, fructose intolerance, Wilson disease, respiratory chain disorders, etc.). The diagnosis is based on highly specific investigations, including crystal identification, biochemical analyses and DNA study. The treatment of nephrolithiasis requires hydration as well as specific measures. Compliance is a major issue regarding the progression of renal damage, but the overall outcome mainly depends on extra-renal involvement in relation to the metabolic defect.

2, 8 Dihydroxyadenine urolithiasis: A case report and review of literature

Adenine phosphoribosyl transferase deficiency is a rare metabolic abnormality presenting with 2,8 dihydroxyadenine urolithiasis. The stones are characteristically radiolucent and therefore need to be differentiated from uric acid stones which are also radiolucent and have identical chemical reactivity. No cases of 2, 8- dihydroxyadenine urolithiasis have been reported from India. We report a 3 year old child with 2, 8- dihydroxyadenine urolithiasis and acute renal failure.

Proteomic analysis of human macrophages exposed to hypochlorite-oxidized low-density lipoprotein

The invasion of monocytes through the endothelial wall of arteries and their transformation from macrophage into form cells has been implicated as a critical initiating event in atherogenesis. Human THP-1 monocytic cells can be induced to differentiate into macrophages by phorbol myristate acetate (PMA) treatment, and can be converted into foam cells by exposure to oxidized low-density lipoprotein (oxLDL). To identify proteins potentially involved in atherosclerotic processes, we performed a proteomic analysis of THP-1 macrophages exposed to oxLDL generated by treatment with native LDL with hypochlorous acid/hypochlorite (HOCl/OCl −). We detected more than a thousand proteins, of which 104 differentially expressed proteins were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF) and the NCBI database. The largest differences in expression were observed for bifunctional purine biosynthesis protein, vacuolar protein sorting 33A, breast carcinoma amplified sequence, adenine phosphoribosyltransferase, and tropomyosin alpha 3 chain. Interestingly, many apoptotic proteins such as lamin B1, poly (ADP-ribose) polymerase, Bcl-2 related protein A1 and vimentin were identified by MALDI–TOF analysis. Identities were confirmed by matching the sequence of several tryptic peptides using MALDI–TOF/TOF MS, Western blot analyses and immunofluorescent microscopy. The data described here will contribute to establishing a functional profile of the human macrophage proteome. Furthermore, the proteins identified in this study are attractive candidates for further biomarkers involved in the pathogenesis of atherosclerosis.

Erythrocytic Adenosine Monophosphate as an Alternative Purine Source in Plasmodium falciparum

Plasmodium falciparum is a purine auxotroph, salvaging purines from erythrocytes for synthesis of RNA and DNA. Hypoxanthine is the key precursor for purine metabolism in Plasmodium. Inhibition of hypoxanthine-forming reactions in both erythrocytes and parasites is lethal to cultured P. falciparum. We observed that high concentrations of adenosine can rescue cultured parasites from purine nucleoside phosphorylase and adenosine deaminase blockade but not when erythrocyte adenosine kinase is also inhibited. P. falciparum lacks adenosine kinase but can salvage AMP synthesized in the erythrocyte cytoplasm to provide purines when both human and Plasmodium purine nucleoside phosphorylases and adenosine deaminases are inhibited. Transport studies in Xenopus laevis oocytes expressing the P. falciparum nucleoside transporter PfNT1 established that this transporter does not transport AMP. These metabolic patterns establish the existence of a novel nucleoside monophosphate transport pathway in P. falciparum.

Leishmania donovani Peroxin 14 Undergoes a Marked Conformational Change following Association with Peroxin 5

The import of PTS1 proteins into the glycosome or peroxisome requires binding of a PTS1-laden PEX5 receptor to the membrane-associated protein PEX14 to facilitate translocation of PTS1 proteins into the lumen of these organelles. Quaternary structure analysis of protozoan parasite Leishmania donovani PEX14 (LdPEX14) revealed that this protein forms a homomeric complex with a size > 670 kDa. Moreover, deletion mapping indicated that disruption of LdPEX14 oligomerization correlated with the elimination of the hydrophobic region and coiled-coil motif present in LdPEX14. Analysis of the LdPEX5-LdPEX14 interaction by isothermal titration calorimetry revealed a molar binding stoichiometry of 1:4 (LdPEX5: LdPEX14) and an in-solution dissociation constant (K(d)) of approximately 74 nm. Calorimetry, circular dichroism, intrinsic fluorescence, and analytical ultracentrifugation experiments showed that binding of LdPEX5 resulted in a dramatic conformational change in the LdPEX14 oligomeric complex that involved the reorganization of the hydrophobic segment in LdPEX14. Finally, limited tryptic proteolysis assays established that in the presence of LdPEX5, LdPEX14 became more susceptible to proteolytic degradation consistent with this protein interaction triggering a significant conformational change in the recombinant and native LdPEX14 structures. These structural changes provide essential clues to how LdPEX14 functions in the translocation of folded proteins across the glycosomal membrane.