Impact of Genetics and Age on Muscle‐type Creatine Kinase

Abstract

Muscle‐type creatine kinase (CKM) plays an important role in physiology by maintaining ATP concentrations. Cells use phosphocreatine as a means to store high energy phosphate. As ATP is rapidly depleted during events such as muscle contraction, creatine kinase enzymes replenish ATP by transferring the phosphate from phosphocreatine to ADP. Additionally, it has been found that CKM has pharmacologically‐relevant activity, as it is involved in the activation of tenofovir (TFV), a nucleotide analog reverse transcriptase inhibitor used for HIV pre‐exposure prophylaxis (PrEP). TFV is a prodrug that requires phosphorylation by intracellular kinases to produce the pharmacologically active tenofovir diphosphate (TFV‐DP) metabolite. In colon tissue, a site of HIV infection, CKM is responsible for the second phosphorylation reaction, converting TFV‐monophosphate (TFV‐MP) to TFV‐DP. While TFV‐based PrEP has been largely successful, some people on PrEP still become infected with HIV. Due to the importance of CKM in TFV activation, we hypothesize that person‐to‐person differences in CKM activity in colon tissue may impact TFV‐DP formation and thus protection from infection. To this extent, we have used a mix of biochemical and mass spectrometry‐based tools to study the impact of both genetics and age on muscle‐type creatine kinase. Wild‐type (WT) human CKM and 15 naturally‐occurring mutants were recombinantly expressed and purified and the impact of these mutations on the activities of the enzyme were determined using in vitro assays. It was found that ten of these mutations led to reductions in TFV‐MP phosphorylation to less than 25% of that of WT. Additionally, only four of these ten mutations resulted in decreases in both the canonical ADP phosphorylation and ATP dephosphorylation reactions: R130H, R132C, W211R, and N286I. Positions 130, 132, and 286 are implicated by protein crystal structures to be involved in substrate binding and phosphoryl transfer. Further biochemical studies using a thermal shift assay and hydrogen‐deuterium exchange mass spectrometry revealed the W211R mutation reduces enzyme activity by impacting the protein structure in a destabilizing manner. In pursuit of understanding the impact of age on CKM, colon tissue from 12‐ and 85‐week‐old C57BL/6J mice (n=5) were analyzed using nano‐liquid chromatography mass spectrometry proteomics. It was found that the abundance of CKM in colon tissue increased by 20% in the older mice compared to the younger comparison group. Additionally, phosphorylation at serine 372 was found in three 12‐week‐old mice and just one of the 85‐week‐old mice. These data suggest that the abundance and post‐translational modification of CKM in colon tissue may change as a function of age, which could have an effect on TFV activation. Together, these data demonstrate that two factors, age and genetics, may lead to interindividual differences in TFV activation, suggesting a potential role for a more personalized approach to PrEP.