The ubiquitous mitochondrial protein unfoldase CLPX regulates erythroid heme synthesis by control of iron utilization and heme synthesis enzyme activation and turnover

Abstract

Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism. Heme plays a critical role in catalyzing life-essential redox reactions in all cells, and its synthesis must be tightly balanced with cellular requirements. Heme synthesis in eukaryotes is tightly regulated by the mitochondrial AAA+ unfoldase CLPX (caseinolytic mitochondrial matrix peptidase chaperone subunit X), which promotes heme synthesis by activation of δ-aminolevulinate synthase (ALAS/Hem1) in yeast and regulates turnover of ALAS1 in human cells. However, the specific mechanisms by which CLPX regulates heme synthesis are unclear. In this study, we interrogated the mechanisms by which CLPX regulates heme synthesis in erythroid cells. Quantitation of enzyme activity and protein degradation showed that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover, rather than its activation. However, we also showed that CLPX is required for PPOX (protoporphyrinogen IX oxidase) activity and maintenance of FECH (ferrochelatase) levels, which are the terminal enzymes in heme synthesis, likely accounting for the heme deficiency and porphyrin accumulation observed in Clpx−/− cells. Lastly, CLPX is required for iron utilization for hemoglobin synthesis during erythroid differentiation. Collectively, our data show that the role of CLPX in yeast ALAS/Hem1 activation is not conserved in vertebrates as vertebrates rely on CLPX to regulate ALAS turnover as well as PPOX and FECH activity. Our studies reveal that CLPX mutations may cause anemia and porphyria via dysregulation of ALAS, FECH, and PPOX activities, as well as of iron metabolism. Heme is a prosthetic group comprising a central iron chelated by a tetrapyrrole ring. It is critical for many life-essential redox processes, such as detoxification, oxygen transport, circadian rhythm, and control of transcription and translation (1Feng D. Lazar M.A. Clocks, metabolism, and the epigenome.Mol. Cell. 2012; 47: 158-167Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar, 2Girvan H.M. Munro A.W. Heme sensor proteins.J. Biol. Chem. 2013; 288: 13194-13203Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 3Martínková M. Kitanishi K. Shimizu T. Heme-based globin-coupled oxygen sensors: Linking oxygen binding to functional regulation of diguanylate cyclase, histidine kinase, and methyl-accepting chemotaxis.J. Biol. Chem. 2013; 288: 27702-27711Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Most of the body's heme is synthesized in differentiating red cells, whose main function is to transport oxygen via hemoglobin (4Chen C. Paw B.H. Cellular and mitochondrial iron homeostasis in vertebrates.Biochim. Biophys. 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Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). CLPX is a ring-shaped homo-hexamer and is best understood for its function in a proteasome-like enzyme complex with the peptidase CLPP (caseinolytic protease proteolytic subunit). The complex of CLPX and CLPP together forms the CLPXP ATP-dependent protease. The CLPXP protease is best understood to facilitate degradation of misfolded mitochondrial proteins. CLPX recognizes specific sequences in protein substrates and unfolds protein tertiary structures through its central pore, presenting the unfolded polypeptide chain to the CLPP proteolytic chamber (10Baker T.A. Sauer R.T. ClpXP, an ATP-powered unfolding and protein-degradation machine.Biochim. Biophys. Acta. 2012; 1823: 15-28Crossref PubMed Scopus (265) Google Scholar, 11Olivares A.O. Baker T.A. Sauer R.T. Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.Nat. Rev. Microbiol. 2016; 14: 33-44Crossref PubMed Scopus (164) Google Scholar). Although CLPXP functions as a protease, detailed studies on yeast and vertebrate cells indicate that CLPX has functions that are distinct from its role in CLPXP. While Clpp−/− mice survive to adulthood (12Gispert S. Parganlija D. Klinkenberg M. Dröse S. Wittig I. Mittelbronn M. Grzmil P. Koob S. Hamann A. Walter M. Büchel F. Adler T. Hrabé de Angelis M. Busch D.H. Zell A. et al.Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.Hum. Mol. Genet. 2013; 22: 4871-4887Crossref PubMed Scopus (112) Google Scholar, 13Jenkinson E.M. Rehman A.U. Walsh T. Clayton-Smith J. Lee K. Morell R.J. Drummond M.C. Khan S.N. Naeem M.A. Rauf B. Billington N. Schultz J.M. Urquhart J.E. Lee M.K. 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Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). from show that CLPX regulate heme synthesis by activation (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google and degradation Y. K. Y. R. K. K. mechanisms for degradation of ALAS1 protein as a of regulation of heme Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google of the ALAS which the of heme ALAS CLPX for its as yeast a CLPP CLPXP not regulate stability in were suggesting that CLPX required for heme synthesis (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). studies that CLPX deficiency cause accumulation of ALAS protein and heme However, erythroid cells an CLPX protein activity for CLPX (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google increased ALAS protein and ALAS activity. The ALAS activity caused resulting from accumulation of IX Y.Y. S. J.R. H. C. Kafina M.D. Z. L. Baker T.A. H. J.D. G. Paw B.H. in human CLPX of synthase and IX to S. A. 2017; PubMed Scopus Google that regulation of heme synthesis by CLPX not conserved to the role of CLPX in erythroid heme regulation and in the role of CLPX in erythroid heme synthesis, we studies in erythroid cells. of yeast ALAS required for CLPX to with and ALAS J.R. J.R. Baker T.A. Mitochondrial ClpX activates an essential enzyme through 2020; PubMed Scopus Google Scholar), to vertebrate ALAS2 we to CLPX required for ALAS2 activation in cells. to the and Clpp in mouse cells and the loss of CLPX and CLPP protein and by and of Clpx−/− and Clpp−/− cells that Clpx−/− cells were with (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. 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PubMed Scopus Google Scholar), we the of Clpp deficiency on with a heme defect in Clpx−/− cells, differentiating Clpx−/− cells a in and Clpx−/− cells also a in data that CLPX is required for heme synthesis, CLPP is and that CLPX regulates heme of its role in the CLPXP proteolytic deficiency also to the the role of in in we a from the of and the role of the gene in that deficiency caused a in erythroid and erythroid cells (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). in by gene we to the roles in The has an which of protein required for embryos the as embryos to degradation embryos were the anemia from erythroid we erythroid cells in N. E. de A. Z. M. R. Zhou Y. Zon L.I. in and is by the Biol. 2012; PubMed Scopus Google Scholar). embryos not erythroid that is not required for erythroid of cells in erythroid cells, such as and In to erythroid cells, cells were and that were caused by in and in is required for erythroid heme synthesis and function is not required for CLPX regulates we the of its and that in Clpx−/− and Clpp−/− cells to cells. ALAS2 in Clpx−/− cells, to cells, in Clpp−/− cells ALAS2 protein increased in both Clpx−/− and Clpp−/− cells data that CLPX and CLPP may regulate ALAS2 protein In to M.C. S. A. P. The mitochondrial complex and Biol. 2016; PubMed Scopus Google Scholar), we not in protein activity in Clpx−/− and Clpp−/− cells CLPXP regulates ALAS2 we ALAS2 turnover in and Clpp−/− cells of protein translation by Y.Y. S. J.R. H. C. Kafina M.D. Z. L. Baker T.A. H. J.D. G. Paw B.H. in human CLPX of synthase and IX to S. A. 2017; PubMed Scopus Google Scholar, is in and its stability is regulated by activation sequences through the PubMed Scopus Google Scholar, Y.Y. interactions between erythroid and protein Biol. Chem. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar). ALAS2 in Clpx−/− and Clpp−/− cells that CLPXP regulates ALAS1 turnover Y. K. Y. R. K. K. mechanisms for degradation of ALAS1 protein as a of regulation of heme Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). ALAS activity showed that Clpx−/− cells increased ALAS that CLPX is not required for ALAS activation Clpp−/− cells ALAS enzyme activity than Clpx−/− cells. in ALAS activity in Clpp−/− cells The of ALAS activity in Clpp−/− to Clpx−/− cells be to in protein stability suggesting that CLPP may play an role in ALAS activation. is that CLPX in Clpp−/− cells CLPX that not in a CLPXP The on ALAS activity not to in as Clpx−/− and Clpp−/− cells the terminal heme to porphyrin in Clpx−/− and Clpp−/− cells in levels, Clpp−/− cells than Clpx−/− cells. in cells, suggesting that CLPXP may regulate heme synthesis of the heme defect in Clpx−/− erythroid cells not a of a in ALAS activation porphyrin CLPX is in the mitochondrial matrix P. J.D. of in cells via 2013; PubMed Scopus Google Scholar), we that CLPX may regulate the matrix terminal heme synthesis PPOX and FECH P. J.D. of in cells via 2013; PubMed Scopus Google mitochondrial iron metabolism Li L. K. C. B. D. Zhou Y. E. A. et is essential for erythroid iron PubMed Scopus Google Scholar). showed that PPOX and FECH protein were in both However, Clpx−/− cells a in PPOX activity to Clpp−/− cells FECH activity in Clpx−/− cells in cells. In differentiating cells, both Clpx−/− and Clpp−/− cells FECH activity Clpp−/− cells not a heme that the FECH deficiency the cause of the heme defect in Clpx−/− cells. also the that CLPX regulated mitochondrial iron the heme defect in Clpx−/− cells with with a iron A.S. Kafina M.D. Huston N.C. Han M. Yien Y.Y. C. J. J.D. et iron transport by a promotes and in 2017; PubMed Scopus Google Scholar). with increased heme in Clpx−/− cells to to that a mitochondrial iron defect to the heme defect in Clpx−/− cells also embryos from with of the mitochondrial iron Li L. K. C. B. D. Zhou Y. E. A. et is essential for erythroid iron PubMed Scopus Google Scholar, A.S. A. In functional and of transport by Biol. Chem. 2018; 293: Full Text Full Text PDF PubMed Scopus Google and as a control for this as anemia by A.S. Kafina M.D. Huston N.C. Han M. Yien Y.Y. C. J. J.D. et iron transport by a promotes and in 2017; PubMed Scopus Google Scholar). the of embryos in were with of embryos from in both and the were not as as the and indicate that iron deficiency plays a role in anemia in the embryos and is not the cause of the Clpx−/− Clpp−/− cells a defect in mitochondrial iron suggesting the defect is not in mitochondrial iron Our indicate that the by which CLPX regulates ALAS in vertebrate cells is by control of its turnover, rather than its activity. also that CLPX is required for PPOX and FECH activity and regulates mitochondrial iron metabolism in differentiating erythroid cells. The porphyrin accumulation observed in these cells is by increased ALAS2 activity in these cells Although the for in heme synthesis is conserved from yeast to in CLPX function The role of CLPX in heme regulation in S. which not a CLPP In CLPX functions as a mitochondrial protein unfoldase that unfolds the of its to ALAS/Hem1 activation (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). a CLPP which forms the CLPXP protease with In vertebrate cells, CLPXP regulates ALAS turnover Y. K. Y. R. K. K. mechanisms for degradation of ALAS1 protein as a of regulation of heme Biol. Chem. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). studies on vertebrate CLPX not the of CLPX for ALAS activity heme Our studies this by the of and Clpp loss of function on the of heme enzymes and ALAS2 as CLPX to be required for erythroid in (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Clpx−/− erythroid cells and ALAS in In to in the heme defect in Clpx−/− erythroid cells not to in ALAS activity and However, ALAS activity in Clpp−/− cells is than that of Clpx−/− cells its protein in Clpx−/− and Clpp−/− cells are that CLPX may play a role in ALAS to Clpp−/− cells a ALAS as CLPX activates in Clpx−/− and Clpp−/− cells, caused by a of with heme synthesis and iron metabolism Clpx−/− cells PPOX and FECH accounting for the heme defect and The in to increased IX caused by and in PPOX and FECH activity. to R. The of IX to activity in mitochondrial of Biol. Chem. Full Text PDF PubMed Google and leads to increased with PPOX activity in Clpx−/− cells. In differentiating cells, FECH activity in Clpp−/− cells, which this not cause heme The heme defect in Clpx−/− cells by an iron which by iron and While CLPX is best for its proteolytic function A.O. Baker T.A. Sauer R.T. Mechanistic insights into bacterial AAA+ proteases and protein-remodelling machines.Nat. Rev. Microbiol. 2016; 14: 33-44Crossref PubMed Scopus (164) Google Scholar), has roles that are distinct from Our data the that CLPX has functions in heme While PPOX protein are in Clpx−/− and Clpp−/− cells, PPOX activity in Clpx−/− cells, suggesting that CLPX plays a role in PPOX activation. iron studies that not erythroid cells an iron metabolism Our studies that CLPX regulates the function of mitochondrial matrix proteins that regulate heme synthesis in erythroid cells. The interactions between CLPX, CLPP, and erythroid heme synthesis are complex and are likely to be and proteins are proteins that are in F. Langer J.D. of mitochondrial CLPXP protease and substrates its central role in 2015; PubMed Scopus Google Scholar). mouse of with a specific showed an in of the CLPP and proteases associated with a in mitochondrial proteins. FECH, and which are in heme synthesis and B. M. S. B. H. C. deficiency of mitochondrial and proteases and loss of mitochondrial J. PubMed Scopus Google Scholar). data with the in FECH activity that we observed in our Clpx−/− and Clpp−/− erythroid the FECH activity may be caused by in iron metabolism in Clpx−/− cells and studies on cells M.C. S. A. P. The mitochondrial complex and Biol. 2016; PubMed Scopus Google Scholar), protein and activity were in Clpx−/− and Clpp−/− cells Lastly, CLPXP with and ALAS1 in the of from ALAS1 heme synthesis is in ALAS2 turnover is not increased during erythroid are increased of heme that CLPXP not the degradation of ALAS2 heme It is likely that the specific roles of CLPXP are regulated by proteins. terminal the function of erythroid heme for hemoglobin synthesis Y.Y. Shi J. Chen C. Cheung J.T.M. Grillo A.S. Shrestha R. Li L. Zhang X. Kafina M.D. Kingsley P.D. King M.J. Ablain J. Li H. Zon L.I. Palis J. et al.FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.J. Biol. Chem. 2018; 293: 19797-19811Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, C. D. Y. Seguin A. Li L. Hildick-Smith G.J. Shah D.I. Cooney J.D. Chen King M.J. Yien Y.Y. Schultz I.J. H. et regulates of the and iron 2013; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Wittig A. M. Dailey T.A. H. Kafina M.D. A.S. Li L. J. et regulates heme 2017; PubMed Scopus Google Scholar, Y.Y. Schultz I.J. Takahashi-Makise N. B. A. G. Li L. Hildick-Smith G.J. Cooney J.D. Pierce E.L. K. Dailey T.A. Miyata N. et is required for erythroid mitochondrial heme PubMed Scopus Google Scholar, J. M. A.W. of in erythroid of mouse by a novel PubMed Scopus Google Scholar). It is that CLPXP regulates heme synthesis in the is not essential for proteins in erythroid cells. Our data on a caused erythroid Y.Y. S. J.R. H. C. Kafina M.D. Z. L. Baker T.A. H. J.D. G. Paw B.H. in human CLPX of synthase and IX to S. A. 2017; PubMed Scopus Google Scholar). activity and with CLPX, the activity of the CLPX that the not ALAS, accumulation of ALAS and of to Our data indicate that CLPX is not required for activation of ALAS, suggesting that be caused by of ALAS also show that CLPX and CLPP are required for FECH activity. The in FECH activity accumulation caused by and may be by an iron CLPX mutations to metabolic in human and (12Gispert S. Parganlija D. Klinkenberg M. Dröse S. Wittig I. Mittelbronn M. Grzmil P. Koob S. Hamann A. Walter M. Büchel F. Adler T. Hrabé de Angelis M. Busch D.H. Zell A. et al.Loss of mitochondrial peptidase Clpp leads to infertility, hearing loss plus growth retardation via accumulation of CLPX, mtDNA and inflammatory factors.Hum. Mol. Genet. 2013; 22: 4871-4887Crossref PubMed Scopus (112) Google Scholar, 13Jenkinson E.M. Rehman A.U. Walsh T. Clayton-Smith J. Lee K. Morell R.J. Drummond M.C. Khan S.N. Naeem M.A. Rauf B. Billington N. Schultz J.M. Urquhart J.E. Lee M.K. Berry A. et al.Perrault syndrome is caused by recessive mutations in CLPP, encoding a mitochondrial ATP-dependent chambered protease.Am. J. Hum. Genet. 2013; 92: 605-613Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, Y.Y. S. J.R. H. C. Kafina M.D. Z. L. Baker T.A. H. J.D. G. Paw B.H. in human CLPX of synthase and IX to S. A. 2017; PubMed Scopus Google Scholar, B. M. S. B. H. C. deficiency of mitochondrial and proteases and loss of mitochondrial J. PubMed Scopus Google Scholar), and CLPX regulates heme synthesis in erythroid cells by control of mitochondrial heme synthesis and iron heme regulation is not to control of ALAS as the also be the terminal the of PPOX and FECH and with iron The of in that CLPX regulates mitochondrial metabolism in a (9Kardon J.R. Yien Y.Y. Huston N.C. Branco D.S. Hildick-Smith G.J. Rhee K.Y. Paw B.H. Baker T.A. Mitochondrial ClpX activates a key enzyme for heme biosynthesis and erythropoiesis.Cell. 2015; 161: 858-867Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, A. Archambault D. Degani R. Iverson E. Tremblay K.D. Mager J. Nuclear-encoded mitochondrial ribosomal proteins are required to initiate gastrulation.Development. 2020; 147dev188714Crossref PubMed Scopus Google Scholar, M.C. S. A. P. The mitochondrial complex and Biol. 2016; PubMed Scopus Google Scholar, F. Langer J.D. of mitochondrial CLPXP protease and substrates its central role in 2015; PubMed Scopus Google Scholar). that CLPX with to mitochondrial metabolism with cellular requirements. the of CLPX function be key for for metabolic and are in the studies were in with and the of data are in the Y.Y. Shi J. Chen C. Cheung J.T.M. Grillo A.S. Shrestha R. Li L. Zhang X. Kafina M.D. Kingsley P.D. King M.J. Ablain J. Li H. Zon L.I. Palis J. et al.FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.J. Biol. Chem. 2018; 293: 19797-19811Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, Y.Y. S. J.R. H. C. Kafina M.D. Z. L. Baker T.A. H. J.D. G. Paw B.H. in human CLPX of synthase and IX to S. A. 2017; PubMed Scopus Google Scholar, A.S. Kafina M.D. Huston N.C. Han M. Yien Y.Y. C. J. J.D. et iron transport by a promotes and in 2017; PubMed Scopus Google Scholar, Y.Y. Schultz I.J. Takahashi-Makise N. B. A. G. Li L. Hildick-Smith G.J. Cooney J.D. Pierce E.L. K. Dailey T.A. Miyata N. et is required for erythroid mitochondrial heme PubMed Scopus Google Scholar). The that of with the of this and for for critical of the and Chen for C. M. M. A. G. H. J. D. and Y. Y. Y. C. M. R. and Y. Y. Y. data C. M. M. A. and Y. Y. Y. C. M. R. and A. D. C. M. M. A. H. S. L. L. G. H. R. J. D. and Y. Y. Y. C. M. M. A. H. S. G. H. R. J. D. and Y. Y. Y. and H. R. and J. D. P. Y. Y. Y. Y. Y. Y. Y. Y. Y. Y. by the Y. and of and a Y. and D. The is the of the and not the of the of

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