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The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

ABSTRACT: NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD⁺ deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cells of the limbs. At birth, Nampt^ΔPrx1 exhibit dramatic limb shortening due to death of growth plate chondrocytes. Administration of the NAD precursor nicotinamide riboside during pregnancy prevents the majority of in utero defects. Depletion of NAD post-birth also promotes chondrocyte death, preventing further endochondral ossification and joint development. In contrast, osteoblast formation still occurs in knockout mice, in line with distinctly different microenvironments and reliance on redox reactions between chondrocytes and osteoblasts. These findings define a critical role for cell-autonomous NAD homeostasis during endochondral bone formation.

SUBMITTER: Warren A

PROVIDER: S-EPMC10276814 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

Warren Aaron A Porter Ryan M RM Reyes-Castro Olivia O Ali Md Mohsin MM Marques-Carvalho Adriana A Kim Ha-Neui HN Gatrell Landon B LB Schipani Ernestina E Nookaew Intawat I O'Brien Charles A CA Morello Roy R Almeida Maria M

Nature communications 20230617 1

NAD is an essential co-factor for cellular energy metabolism and multiple other processes. Systemic NAD<sup>+</sup> deficiency has been implicated in skeletal deformities during development in both humans and mice. NAD levels are maintained by multiple synthetic pathways but which ones are important in bone forming cells is unknown. Here, we generate mice with deletion of Nicotinamide Phosphoribosyltransferase (Nampt), a critical enzyme in the NAD salvage pathway, in all mesenchymal lineage cell ...[more]

PMID: 37330524

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Project description:The genome of T4-type Vibrio bacteriophage KVP40 has five genes predicted to encode proteins of pyridine nucleotide metabolism, of which two, nadV and natV, would suffice for an NAD+ salvage pathway. NadV is an apparent nicotinamide phosphoribosyltransferase (NAmPRTase), and NatV is an apparent bifunctional nicotinamide mononucleotide adenylyltransferase (NMNATase) and nicotinamide-adenine dinucleotide pyrophosphatase (Nudix hydrolase). Genes encoding the predicted salvage pathway were cloned and expressed in Escherichia coli, the proteins were purified, and their enzymatic properties were examined. KVP40 NadV NAmPRTase is active in vitro, and a clone complements a Salmonella mutant defective in both the bacterial de novo and salvage pathways. Similar to other NAmPRTases, the KVP40 enzyme displayed ATPase activity indicative of energy coupling in the reaction mechanism. The NatV NMNATase activity was measured in a coupled reaction system demonstrating NAD+ biosynthesis from nicotinamide, phosphoribosyl pyrophosphate, and ATP. The NatV Nudix hydrolase domain was also shown to be active, with preferred substrates of ADP-ribose, NAD+, and NADH. Expression analysis using reverse transcription-quantitative PCR (qRT-PCR) and enzyme assays of infected Vibrio parahaemolyticus cells demonstrated nadV and natV transcription during the early and delayed-early periods of infection when other KVP40 genes of nucleotide precursor metabolism are expressed. The distribution and phylogeny of NadV and NatV proteins among several large double-stranded DNA (dsDNA) myophages, and also those from some very large siphophages, suggest broad relevance of pyridine nucleotide scavenging in virus-infected cells. NAD+ biosynthesis presents another important metabolic resource control point by large, rapidly replicating dsDNA bacteriophages.IMPORTANCE T4-type bacteriophages enhance DNA precursor synthesis through reductive reactions that use NADH/NADPH as the electron donor and NAD+ for ADP-ribosylation of proteins involved in transcribing and translating the phage genome. We show here that phage KVP40 encodes a functional pyridine nucleotide scavenging pathway that is expressed during the metabolic period of the infection cycle. The pathway is conserved in other large, dsDNA phages in which the two genes, nadV and natV, share an evolutionary history in their respective phage-host group.

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The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

Publications

The NAD salvage pathway in mesenchymal cells is indispensable for skeletal development in mice.

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