Related Experiment Videos
In vitro phosphorylation of cytoskeletal proteins in the rat cerebral cortex is decreased by propionic acid
A de Mattos-Dutra1, M Sampaio de Freitas, N Schröder
1Departamento de Bioquímica, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil.
Insights
Propionic acid (PA) inhibits key enzymes involved in protein phosphorylation, including cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1). This disruption of cytoskeletal protein phosphorylation may contribute to neurological dysfunction in propionic acidemia.
Area of Science:
- Biochemistry
- Neuroscience
- Metabolic Disorders
Background:
- Propionic acidemia is a metabolic disorder characterized by the accumulation of propionic acid (PA).
- Neurological dysfunction is a common symptom in patients with propionic acidemia.
- Cytoskeletal proteins, such as neurofilaments and tubulin, are crucial for neuronal structure and function.
Purpose of the Study:
- To investigate the in vitro effects of propionic acid on the phosphorylation of cytoskeletal proteins.
- To determine if propionic acid affects the activity of key enzymes involved in protein phosphorylation and dephosphorylation.
Main Methods:
- Assayed the in vitro incorporation of [32P]ATP into neurofilament subunits and tubulin.
- Investigated the effects of propionic acid on cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1) activities.
- Utilized specific activators and inhibitors for the studied enzymes.
Main Results:
- Propionic acid decreased [32P]ATP incorporation into neurofilament subunits (NF-M, NF-L) and tubulin.
- Propionic acid inhibited the stimulatory effects of cAMP on PKA and interfered with its inhibitor PKAI.
- Propionic acid partially prevented the stimulatory effect of Ca2+/calmodulin on CaMKII and interfered with its inhibitor KN-93.
- Propionic acid totally inhibited PP1-mediated dephosphorylation of neurofilaments and tubulin.
Conclusions:
- Propionic acid inhibits the in vitro activities of PKA, CaMKII, and PP1 in the rat cerebral cortex cytoskeletal fraction.
- Altered phosphorylation of cytoskeletal proteins by propionic acid may contribute to the neurological dysfunction observed in propionic acidemia.
Abstract:
In the present study we demonstrate that propionic acid (PA), a metabolite that accumulates in large amounts in propionic acidemia, is able to decrease in vitro incorporation of [32P]ATP into neurofilament subunits (NF-M and NF-L) and alpha- and beta-tubulin. Considering that the endogenous phosphorylating system associated with the cytoskeletal fraction contains cAMP-dependent protein kinase (PKA), Ca2+/calmodulin protein kinase II (CaMKII), and protein phosphatase 1 (PP1), we first assayed the effect of the acid on the kinase activities by using the specific activators cAMP and Ca2+/calmodulin or the inhibitors PKAI or KN-93 for PKA and CaMKII, respectively. Results demonstrated that the acid totally inhibited the stimulatory effect of cAMP and interfered with the inhibitory effect of PKAI. In addition, PA partially prevented the stimulatory effect of Ca2+/calmodulin and interfered with the effect of KN-93. In addition, we demonstrated that PA totally inhibited in vitro dephosphorylation of neurofilament subunits and tubulins mediated by PP1 in brain slices pretreated with the acid. Taken together, these results demonstrate that PA inhibits the in vitro activities of PKA, CaMKII, and PP1 associated with the cytoskeletal fraction of the cerebral cortex of rats. This study suggests that PA at the same concentrations found in tissues from propionic acidemic children may alter phosphorylation of cytoskeletal proteins, which may contribute to the neurological dysfunction characteristic of propionic acidemia.