Page 8 - Read Online

Page 219 Mejia et al. J Transl Genet Genom 2024;8:216-24 https://dx.doi.org/10.20517/jtgg.2024.11

Figure 1. CL levels are reduced and trioleoyl-MLCL and citrate synthase activity elevated in BTHS lymphoblasts. Quantification of the
major CL. (A) and Major MLCL (B) Fatty acyl molecular species in age-matched control and BTHS lymphoblasts as described in
Materials and Methods. (C) Mitochondrial fractions were prepared from age-matched control and BTHS lymphoblasts and citrate
#
synthase activity determined as described in Materials and Methods. Data represent the mean + SD, n = 4. P < 0.001; **P < 0.01;
*P < 0.05; ns: not significant.

We previously observed that PKCδ phosphorylation was altered on several sites examined in BTHS
lymphoblasts . Since altered phosphorylation may affect PKCδ activation , we examined if this was
[13]
[15]
related to altered PKCδ associated with higher molecular weight complex in mitochondria of BTHS patient
lymphoblasts. Mitochondrial fractions were subjected to BN-PAGE followed by immunoblot analysis for
determination of PKCδ levels. The two upper bands indicated on the left of the blot are molecular mass
markers at 1,236 and 1,048 kDa, respectively [Figure 2A]. The level of PKCδ located on the gel at a predicted
molecular mass near 77.5 kDa was elevated by 1.5-fold (P < 0.05) in BTHS lymphoblasts compared to age-
matched control cells. In contrast, the level of PKCδ located on the gel at approximately 480 kDa was
reduced by 72% (P < 0.01) in BTHS lymphoblasts compared to age-matched control cells [Figure 2B]. Thus,
BTHS lymphoblasts exhibit elevated expression of PKCδ but reduced PKCδ associated with a higher
molecular weight complex in mitochondria.

Since oxidative phosphorylation is impaired in BTHS B lymphoblasts , it is possible that enhanced creatine
[9]
uptake may occur as a compensatory mechanism to maintain energy metabolism as observed with other
[17]
metabolites such as glucose . Control and BTHS lymphoblasts were incubated with [ C]Creatine for up to
14
60 min and radioactivity incorporated into cells determined. [ C]Creatine incorporation into BTHS
14
lymphoblasts was markedly elevated compared to control cells [Figure 3]. Thus, BTHS lymphoblasts exhibit
enhanced creatine uptake.

DISCUSSION
BTHS is a rare X-linked genetic disease and is the only known disease in which the specific biochemical
defect is a reduction in CL and accumulation of MLCL . We observed a reduction in all major molecular
[1-3]
species of CL in BTHS lymphoblasts accompanied by a > 20-fold elevation in trioleoyl-MLCL. Previous
studies demonstrated an increase in abnormal mitochondrial mass in BTHS patient lymphoblasts . We
[7,8]