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Role for Cytoplasmic Nucleotide Hydrolysis in Hepatic Function and Protein Synthesis

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Role for Cytoplasmic Nucleotide Hydrolysis in Hepatic Function and Protein Synthesis Role for cytoplasmic nucleotide hydrolysis in hepatic function and protein synthesis Benjamin H. Hudson1, Joshua P. Frederick1, Li Yin Drake, Louis C. Megosh, Ryan P. Irving, and John D. York2,3 Department of Pharmacology and Cancer Biology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC 27710 Edited by David W. Russell, University of Texas Southwestern Medical Center, Dallas, TX, and approved February 11, 2013 (received for review March 26, 2012) Nucleotide hydrolysis is essential for many aspects of cellular (5–7), which is degraded to 5′-AMP by a family of enzymes known function. In the case of 3′,5′-bisphosphorylated nucleotides, mam- as 3′-nucleotidases (8). mals possess two related 3′-nucleotidases, Golgi-resident 3′-phos- Mammalian genomes encode two 3′-nucleotidases, the recently phoadenosine 5′-phosphate (PAP) phosphatase (gPAPP) and characterized Golgi-resident PAP phosphatase (gPAPP) and Bisphosphate 3′-nucleotidase 1 (Bpnt1). gPAPP and Bpnt1 localize Bisphosphate 3′-nucleotidase 1 (Bpnt1), which localize to the to distinct subcellular compartments and are members of a con- Golgi lumen and cytoplasm, respectively (Fig. 1B)(8–11). gPAPP served family of metal-dependent lithium-sensitive enzymes. Al- and Bpnt1 are members of a family of small-molecule phospha- though recent studies have demonstrated the importance of tases whose activities are both dependent on divalent cations and gPAPP for proper skeletal development in mice and humans, the inhibited by lithium (8). The family comprises seven mammalian role of Bpnt1 in mammals remains largely unknown. Here we re- gene products: fructose bisphosphatase 1 and 2, inositol monophos- port that mice deficient for Bpnt1 do not exhibit skeletal defects phatase 1 and 2, inositol polyphosphate 1-phosphatase, gPAPP, but instead develop severe liver pathologies, including hypopro- and Bpnt1 (Fig. 1A). Although the members display limited overall teinemia, hepatocellular damage, and in severe cases, frank whole- sequence similarity, their shared properties are defined by a com- body edema and death. Accompanying these phenotypes, we ob- mon structural core and the catalytic motif, D-Xn-EE-Xn-DP(i/l) served tissue-specific elevations of the substrate PAP, up to 50-fold D(s/g/a)T-Xn-WDXn-11GG (12). in liver, repressed translation, and aberrant nucleolar architecture. Although Bpnt1 and gPAPP share a common substrate, their Remarkably, the phenotypes of the Bpnt1 knockout are rescued by localization to distinct cellular compartments suggests that they fi generating a double mutant mouse de cient for both PAP synthe- play unique roles in the cell. gPAPP, whose evolution parallels the sis and hydrolysis, consistent with a mechanism in which PAP appearance of metazoan-specific Golgi-localized sulfation, is es- accumulation is toxic to tissue function independent of sulfation. fi sential for normal development. Its inactivation in mice results in Overall, our study de nes a role for Bpnt1 in mammalian physi- pulmonary insufficiency, joint defects, and impaired skeletal de- ology and provides mechanistic insights into the importance of velopment due to inadequate chondroitin and heparan sulfation sulfur assimilation and cytoplasmic PAP hydrolysis to normal (11, 13). In addition, the identification of a phenotypically related liver function. human skeletal disease resulting from mutations in gPAPP con- firms that Golgi-resident 3′-nucleotidase activity is essential for ribosome biogenesis | nucleolus | phosphoadenosine phosphosulfate | sulfation-dependent processes such as cartilage formation and exoribonuclease long bone growth (14). Conversely, functional studies in bacteria and yeast have implicated the more ancient cytosolic 3′-nucleo- he liver is an important hub for a diverse array of processes, fi tidase Bpnt1 in numerous sulfation-independent processes, in- Tincluding metabolism, detoxi cation, and protein production. cluding salt tolerance and methionine biosynthesis (15–23). Thus, To meet these demands, hepatocytes, the fundamental units of although it is clear that gPAPP is essential for proper Golgi- the liver that account for 70% of its weight, perform hundreds of mediated sulfation, the role of Bpnt1 in mammals and its po- different biochemical reactions. One component of hepatocel- tential connection to cytosolic sulfation remains unclear. lular function that is essential for numerous downstream physi- Here we report the generation and analysis of Bpnt1 global ological processes is the synthesis and export of more than 100 knockout mice. Loss of cytoplasmic 3′-nulceotidase function plasma proteins (1, 2). Albumin, which accounts for greater than results in markedly distinct phenotypes compared with those 60% of the hepatically produced plasma protein content, trans- observed in gPAPP- or SULT-deficient animals. Our data pro- ports hormones, fatty acids, metals, and xenobiotics throughout vide an unanticipated genetic basis for pathologies that in severe the body and provides roughly 75% of the vascular osmotic pres- cases lead to liver failure, whole-body edema, and death and sure (2). Because of its importance, mammals have developed whose etiology seems to be independent of sulfation. Addition- multiple safeguards to maintain consistent serum albumin con- fi ally, we demonstrate a genetic strategy to completely reverse the centrations, and as a result, signi cant reductions in its abundance physiological defects in Bpnt1-deficient mice, which may inspire are generally only seen in chronic liver diseases such as cirrhosis. Indeed, loss of oncotic pressure and concurrent presentation of edema is often indicative of liver failure (1). fi Author contributions: B.H.H., J.P.F., L.Y.D., and J.D.Y. designed research; B.H.H., J.P.F., The liver also provides the rst layer of defense against cyto- L.Y.D., L.C.M., and R.P.I. performed research; B.H.H., J.P.F., and L.Y.D. contributed new toxic agents through a number of biotransformation pathways reagents/analytic tools; B.H.H., J.P.F., and J.D.Y. analyzed data; and B.H.H. and J.D.Y. (3, 4), one of which is mediated by the sulfotransferase (SULT) wrote the paper. superfamily of enzymes that catalyzes the transfer of activated The authors declare no conflict of interest. sulfur from the universal donor 3′-phosphoadenosine 5′-phos- This article is a PNAS Direct Submission. phosulfate (PAPS) to acceptor molecules. PAPS is synthesized Freely available online through the PNAS open access option. from inorganic sulfate through the successive sulfurylase and 1B.H.H. and J.P.F. contributed equally to this work. kinase activities of PAPS synthases, such as mammalian Papss2 2Present address: Department of Biochemistry, Vanderbilt University Medical Center, (5–8), and although PAPS in metazoans is consumed exclusively Nashville, TN 37232-0146. by SULTs, its uses diverge widely in other kingdoms of life 3To whom correspondence should be addressed. E-mail: [email protected]. (8). Regardless of the acceptor molecule, PAPS-using reactions This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. generate the byproduct 3′-phosphoadenosine 5′-phosphate (PAP) 1073/pnas.1205001110/-/DCSupplemental. 5040–5045 | PNAS | March 26, 2013 | vol. 110 | no. 13 www.pnas.org/cgi/doi/10.1073/pnas.1205001110 Downloaded by guest on September 26, 2021 A SDS/PAGE and clinical chemistries. Strikingly, Bpnt1 null mice showed significantly lower levels of many serum proteins com- pared with wild types (Fig. 2B). One protein we found to be Phosphatase Subcellular Preferred markedly repressed, albumin, normally constitutes greater than family member localization substrate 60% of the total serum protein content and provides the principle Fructose bisphosphatase 1/2 Fructose component of the osmotic pressure necessary to maintain fluid (Fbp1/2) Cytosol 1,6-bisphosphate Inositol monophosphatase 1/2 Inositol (Impa1/2) Cytosol monophosphate A wild-type Bpnt1-/- Inositol polyphosphate 1-phosphatase Inositol Cytosol (Inpp1) polyphosphate Golgi-resident PAP phosphatase 3’-phosphoadenosine Gogli apparatus (gPAPP) 5’-phosphate Bisphosphate 3’nucleotidase 1 3’-phosphoadenosine Cytosol (Bpnt1) 5’-phosphate +/+ -/- B edema C 2- cytosol Golgi 4.0 B SO ATP lumen 4 250 ATP sulfurylase 150 Papss2 3.0 5’ APS ATP 100 APS 75 kinase 2.0 PAPS PAPS PAPS 50 R OH transporter R OH cytosolic Golgi 37 R S SULTs SULTs R S 1.0 PAP PAP 25 Serum Albumin (g/dL) 15 Bpnt1 gPAPP 0.0 +/+ +/- -/- -/- 5’ AMP 5’ AMP serum coomassie edema Fig. 1. A lithium-sensitive family of small-molecule phosphatases and the D +/- -/- +/- -/- sulfate assimilation pathway. (A)TheMus Musculus family of metal-dependent/ lithium-sensitive phosphomonoesterases and the consensus core motif that defines the family. (B) Schematic of the sulfate assimilation pathway inter- 75 mediates. Bpnt1 and gPAPP hydrolyze the 3′ phosphate from the sulfation byproduct PAP in the cytoplasm and Golgi lumen, respectively. In mice, Papps2 encodes a dual functional enzyme with both ATP sulfurylase and APS 50 kinase activities that synthesize PAPS from sulfate and ATP. 37 pharmacological approaches to overcoming 3′-nucleotidase de- ficiency. Our study provides insights into the role of Bpnt1 in 25 mammalian physiology and illuminates the unique contributions fi ′ coomassie autoradiograph of compartment-speci c3-nucleotide hydrolysis. E -/- Results 3.0 80S wild-type 3.0 80S Bpnt1 2.5 2.5 CELL BIOLOGY Bpnt1 Null Mice Develop Edema as a Result of Liver Dysfunction. To 2.0 40S 2.0 40S further our understanding of Bpnt1’s role in mammals we used 1.5 Polysomes 1.5 1.0 1.0 60S homologous recombination to generate global knockout mice 60S A SI Appendix 0.5 0.5 Polysomes (Fig. 2 and , Fig. S1). Heterozygous and homozygous 0.0 0.0 null animals were viable and developed according to expected Relavtive A260 -0.5 -0.5 Mendelian distributions. Analysis of tissues from homozygous -1.0 -1.0 null animals by Western blot revealed a complete absence of sucrose density sucrose density detectable Bpnt1 (SI Appendix, Fig. S2). At birth and through early development, homozygous null mice appear grossly identi- Fig. 2.
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