Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail residues, Ser-104 and Ser-116, allosterically mediates conformational changes of the DED and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas-associated death domain (FADD) protein. We delineated that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and between the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail residues of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong electrostatic interactions with arginine residues on FADD DED. Upon PEA-15 binding, FADD repositions its death domain (DD) relative to the DED, an essential conformational change to allow the death-inducing signaling complex (DISC) assembly.

PEA‐15, phosphoprotein enriched in astrocytes, 15 kDa, performs a variety of functions in regulating cellular pathways, such as cell proliferation and apoptosis. The protein consists of a death effector domain (DED) and a long, irregular structured C‐terminal tail. We hypothesize that phosphorylation of the C‐terminal tail residues, Ser‐104 and Ser‐116, promotes conformational changes at the DED, and alters the binding specificity from extracellular regulated kinase‐2 (ERK2) to Fas associated death domain (FADD). To test the hypothesis, we used the molecular dynamics package, GROMACS, to simulate the complexes between unphosophorylated PEA‐15 and ERK2, and doubly phosphorylated PEA‐15 (PEA‐15pp) and FADD on a GPU‐equipped Linux workstation, with simulation time up to 150 ns. The simulated complex structures were analyzed and visualized with VMD and PyMOL programs. The computational experiments revealed that the DED conformations and surface polar interactions are dependent on phosphorylation states of the C‐terminal serine residues. The binding interfaces between PEA‐15 and ERK and PEA‐15pp and FADD are similarly composed of a scaffold that includes both DED and C‐terminal tail residues of PEA‐15. Residues from helices 5 and 6 of the DED directly interact with either ERK2 or FADD, while helices 2, 3, and 4 become more flexible in the complex structures than the free‐form protein. PEA‐15 also uses the same stretch of its irregular C‐terminal tail to interact with FADD or ERK2 (residues 116‐130). Phosphorylation of Ser‐104 and Ser‐116 on the C‐terminal tail alters the binding specificity from ERK2 to FADD, as the negatively charged phosphoryl groups interacts with positively charged amino acids on FADD, while there are no charge‐charge interactions of the C‐terminal tail with ERK2.

Phosphoprotein enriched in astrocytes, 15 kDa (PEA-15) is a death-effector domain (DED) containing protein involved in regulating mitogen-activated protein kinase and apoptosis pathways. In this molecular-dynamics study, we examined how phosphorylation of the PEA-15 C-terminal tail Ser-104 and Ser-116 allosterically promotes conformational changes of the DED, and alters the binding specificity from extracellular-regulated kinase (ERK) to Fas associated death domain (FADD) protein. We found that the binding interfaces between the unphosphorylated PEA-15 and ERK2 and the doubly phosphorylated PEA-15 and FADD are similarly composed of a scaffold that includes both the DED and the C-terminal tail of PEA-15. While the unphosphorylated serine residues do not directly interact with ERK2, the phosphorylated Ser-116 engages in strong interactions with arginine residues on FADD DED. In this DED complex, FADD repositions its death domain (DD) relative to the DED, which has strong implications on the association of the death-inducing signaling complex (DISC).

Human brain microvascular endothelial cells (hBMVEC), together with astrocytes and pericytes, construct the blood‐brain barrier (BBB) to protect the brain from toxins and pathogens via paracellular, transcellular, transporter, and extracellular matrices, and tight junction proteins. The BBB also inhibits many therapeutic substances from entering the brain, making it challenging to design drugs that can migrate through the BBB effectively. The objective of the project was to understand the detrimental effects of psychological stimulants (cocaine, THC, Amphetamine) and HIV Tat proteins on the integrity of the BBB, and to inform the potential therapeutic strategies. The hBMVEC cell culture was treated with the stimulants and two different constructs of HIV Tat proteins. Western blot and RT‐qPCR techniques were used to detect pyroptosis and apoptosis proteins that could lead to endothelial cell death. We found that proteins involved in apoptosis (PEA‐15, caspase‐3, and caspase‐8) were more expressed and upregulated comparing to proteins involved in pyroptosis (caspase‐1, gasdermin D) and cytokine proteins (IL‐1β, IL‐6, IL‐18).

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