Structural and dynamic effects of paraoxon binding to human acetylcholinesterase by X-ray crystallography and inelastic neutron scattering.

Organophosphorus (OP) compounds, including nerve agents and some pesticides, covalently bind to the catalytic serine of human acetylcholinesterase (hAChE), thereby inhibiting acetylcholine hydrolysis necessary for efficient neurotransmission. Oxime antidotes can reactivate the OP-conjugated hAChE, but reactivation efficiency can be low for pesticides, such as paraoxon (POX). Understanding structural and dynamic determinants of OP inhibition and reactivation can provide insights to design improved reactivators. Here, X-ray structures of hAChE with unaged POX, with POX and oximes MMB4 and RS170B, and with MMB4 are reported. A significant conformational distortion of the acyl loop was observed upon POX binding, being partially restored to the native conformation by oximes. Neutron vibrational spectroscopy combined with molecular dynamics simulations showed that picosecond vibrational dynamics of the acyl loop soften in the ∼20-50 cm-1 frequency range. The acyl loop structural perturbations may be correlated with its picosecond vibrational dynamics to yield more comprehensive template for structure-based reactivator design.

Covalent inhibition of hAChE by organophosphates causes homodimer dissociation through long-range allosteric effects.

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.

Defective internal allosteric network imparts dysfunctional ATP/substrate-binding cooperativity in oncogenic chimera of protein kinase A.

An aberrant fusion of the DNAJB1 and PRKACA genes generates a chimeric protein kinase (PKA-CDNAJB1) in which the J-domain of the heat shock protein 40 is fused to the catalytic α subunit of cAMP-dependent protein kinase A (PKA-C). Deceivingly, this chimeric construct appears to be fully functional, as it phosphorylates canonical substrates, forms holoenzymes, responds to cAMP activation, and recognizes the endogenous inhibitor PKI. Nonetheless, PKA-CDNAJB1 has been recognized as the primary driver of fibrolamellar hepatocellular carcinoma and is implicated in other neoplasms for which the molecular mechanisms remain elusive. Here we determined the chimera's allosteric response to nucleotide and pseudo-substrate binding. We found that the fusion of the dynamic J-domain to PKA-C disrupts the internal allosteric network, causing dramatic attenuation of the nucleotide/PKI binding cooperativity. Our findings suggest that the reduced allosteric cooperativity exhibited by PKA-CDNAJB1 alters specific recognitions and interactions between substrates and regulatory partners contributing to dysregulation.

Multi-state recognition pathway of the intrinsically disordered protein kinase inhibitor by protein kinase A.

In the nucleus, the spatiotemporal regulation of the catalytic subunit of cAMP-dependent protein kinase A (PKA-C) is orchestrated by an intrinsically disordered protein kinase inhibitor, PKI, which recruits the CRM1/RanGTP nuclear exporting complex. How the PKA-C/PKI complex assembles and recognizes CRM1/RanGTP is not well understood. Using NMR, SAXS, fluorescence, metadynamics, and Markov model analysis, we determined the multi-state recognition pathway for PKI. After a fast binding step in which PKA-C selects PKI's most competent conformations, PKI folds upon binding through a slow conformational rearrangement within the enzyme's binding pocket. The high-affinity and pseudo-substrate regions of PKI become more structured and the transient interactions with the kinase augment the helical content of the nuclear export sequence, which is then poised to recruit the CRM1/RanGTP complex for nuclear translocation. The multistate binding mechanism featured by PKA-C/PKI complex represents a paradigm on how disordered, ancillary proteins (or protein domains) are able to operate multiple functions such as inhibiting the kinase while recruiting other regulatory proteins for nuclear export.

A Mixed Methods Approach to Assess the Impact of an Interprofessional Education Medical Error Simulation.

Objective. To evaluate the impact of a single, half-day interprofessional education (IPE) simulation on disclosing medical errors, and to compare the impacts on pharmacy students with those on students from other health professional programs. Methods. A mixed methods approach was used to provide a comprehensive understanding of the immediate and persistent outcomes of a realistic medical error disclosure simulation. Anonymous pre- and post-simulation quantitative data were collected using a validated attitudinal survey instrument administered at the time of the simulation. To assess more sustained impacts of the simulation, uni-professional focus groups were held several months following the simulation. Results. The survey analysis showed that a significant positive change in attitudes towards teamwork, roles and responsibilities occurred in students in most of the professions represented, with pharmacy students experiencing positive changes across a wider range of interprofessional attitudes. The focus group results showed that there were persistent impacts across all professions on learners' knowledge, skills, attitudes, and confidence in disclosing medical errors in interprofessional teams. Conclusion. Mixed methods analysis of a high-fidelity IPE error disclosure simulation demonstrated that single IPE activities, if realistic, can have significant positive impacts on students' interprofessional attitudes and competencies, and increase confidence in conducting team-based error disclosures.

Rational design, synthesis, and evaluation of uncharged, "smart" bis-oxime antidotes of organophosphate-inhibited human acetylcholinesterase.

Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake of OPs into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are nonproductive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE, here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-human AChE conjugates. Oxime group reorientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.

Productive reorientation of a bound oxime reactivator revealed in room temperature X-ray structures of native and VX-inhibited human acetylcholinesterase.

Exposure to organophosphorus compounds (OPs) may be fatal if untreated, and a clear and present danger posed by nerve agent OPs has become palpable in recent years. OPs inactivate acetylcholinesterase (AChE) by covalently modifying its catalytic serine. Inhibited AChE cannot hydrolyze the neurotransmitter acetylcholine leading to its build-up at the cholinergic synapses and creating an acute cholinergic crisis. Current antidotes, including oxime reactivators that attack the OP-AChE conjugate to free the active enzyme, are inefficient. Better reactivators are sought, but their design is hampered by a conformationally rigid portrait of AChE extracted exclusively from 100K X-ray crystallography and scarcity of structural knowledge on human AChE (hAChE). Here, we present room temperature X-ray structures of native and VX-phosphonylated hAChE with an imidazole-based oxime reactivator, RS-170B. We discovered that inhibition with VX triggers substantial conformational changes in bound RS-170B from a "nonproductive" pose (the reactive aldoxime group points away from the VX-bound serine) in the reactivator-only complex to a "semi-productive" orientation in the VX-modified complex. This observation, supported by concurrent molecular simulations, suggested that the narrow active-site gorge of hAChE may be significantly more dynamic than previously thought, allowing RS-170B to reorient inside the gorge. Furthermore, we found that small molecules can bind in the choline-binding site hindering approach to the phosphorous of VX-bound serine. Our results provide structural and mechanistic perspectives on the reactivation of OP-inhibited hAChE and demonstrate that structural studies at physiologically relevant temperatures can deliver previously overlooked insights applicable for designing next-generation antidotes.

Differential gene expression patterns in vein regions susceptible versus resistant to neointimal hyperplasia.

Arteriovenous hemodialysis graft (AVG) stenosis results in thrombosis and AVG failure, but prevention of stenosis has been unsuccessful due in large part to our limited understanding of the molecular processes involved in neointimal hyperplasia (NH) formation. AVG stenosis develops chiefly as a consequence of highly localized NH formation in the vein-graft anastomosis region. Surprisingly, the vein region just downstream of the vein-graft anastomosis (herein termed proximal vein region) is relatively resistant to NH. We hypothesized that the gene expression profiles of the NH-prone and NH-resistant regions will be different from each other after graft placement, and analysis of their genomic profiles may yield potential therapeutic targets to prevent AVG stenosis. To test this, we evaluated the vein-graft anastomosis (NH-prone) and proximal vein (NH-resistant) regions in a porcine model of AVG stenosis with a porcine microarray. Gene expression changes in these two distinct vein regions, relative to the gene expression in unoperated control veins, were examined at early (5 days) and later (14 days) time points following graft placement. Global genomic changes were much greater in the NH-prone region than in the NH-resistant region at both time points. In the NH-prone region, genes related to regulation of cell proliferation and osteo-/chondrogenic vascular remodeling were most enriched among the significantly upregulated genes, and genes related to smooth muscle phenotype were significantly downregulated. These results provide insights into the spatial and temporal genomic modulation underlying NH formation in AVG and suggest potential therapeutic strategies to prevent and/or limit AVG stenosis.

An N-terminally truncated form of cyclic GMP-dependent protein kinase Iα (PKG Iα) is monomeric and autoinhibited and provides a model for activation.

The type I cGMP-dependent protein kinases (PKG I) serve essential physiological functions, including smooth muscle relaxation, cardiac remodeling, and platelet aggregation. These enzymes form homodimers through their N-terminal dimerization domains, a feature implicated in regulating their cooperative activation. Previous investigations into the activation mechanisms of PKG I isoforms have been largely influenced by structures of the cAMP-dependent protein kinase (PKA). Here, we examined PKG Iα activation by cGMP and cAMP by engineering a monomeric form that lacks N-terminal residues 1-53 (Δ53). We found that the construct exists as a monomer as assessed by whole-protein MS, size-exclusion chromatography, and small-angle X-ray scattering (SAXS). Reconstruction of the SAXS 3D envelope indicates that Δ53 has a similar shape to the heterodimeric RIα-C complex of PKA. Moreover, we found that the Δ53 construct is autoinhibited in its cGMP-free state and can bind to and be activated by cGMP in a manner similar to full-length PKG Iα as assessed by surface plasmon resonance (SPR) spectroscopy. However, we found that the Δ53 variant does not exhibit cooperative activation, and its cyclic nucleotide selectivity is diminished. These findings support a model in which, despite structural similarities, PKG Iα activation is distinct from that of PKA, and its cooperativity is driven by in trans interactions between protomers.

Structure of a PKA RIα Recurrent Acrodysostosis Mutant Explains Defective cAMP-Dependent Activation.

Most disease-related mutations that impair cAMP protein kinase A (PKA) signaling are present within the regulatory (R) PKA RI alpha-subunit (RIα). Although mutations in the PRKAR1A gene are linked to Carney complex (CNC) disease and, more recently, to acrodysostosis-1 (ACRDYS1), the two diseases show contrasting phenotypes. While CNC mutations cause increased PKA activity, ACRDYS1 mutations result in decreased PKA activity and cAMP resistant holoenzymes. Mapping the ACRDYS1 disease mutations reveals their localization to the second of two tandem cAMP-binding (CNB) domains (CNB-B), and here, we characterize a recurrent deletion mutant where the last 14 residues are missing. The crystal structure of a monomeric form of this mutant (RIα92-365) bound to the catalytic (C)-subunit reveals the dysfunctional regions of the RIα subunit. Beyond the missing residues, the entire capping motif is disordered (residues 357-379) and explains the disrupted cAMP binding. Moreover, the effects of the mutation extend far beyond the CNB-B domain and include the active site and N-lobe of the C-subunit, which is in a partially open conformation with the C-tail disordered. A key residue that contributes to this crosstalk, D267, is altered in our structure, and we confirmed its functional importance by mutagenesis. In particular, the D267 interaction with Arg241, a residue shown earlier to be important for allosteric regulation, is disrupted, thereby strengthening the interaction of D267 with the C-subunit residue Arg194 at the R:C interface. We see here how the switch between active (cAMP-bound) and inactive (holoenzyme) conformations is perturbed and how the dynamically controlled crosstalk between the helical domains of the two CNB domains is necessary for the functional regulation of PKA activity.

Limitations in current acetylcholinesterase structure-based design of oxime antidotes for organophosphate poisoning.

Acetylcholinesterase (AChE; EC 3.1.1.7), an essential enzyme of cholinergic neurotransmission in vertebrates, is a primary target in acute nerve agent and organophosphate (OP) pesticide intoxication. Catalytically inactive OP-AChE conjugates formed between the active-center serine and phosphorus of OPs can, in principle, be reactivated by nucleophilic oxime antidotes. Antidote efficacy is limited by the structural diversity of OP-AChE conjugates resulting from differences in the structure of the conjugated OP, the different active-center volumes they occupy when conjugated to the active-center serine of AChE, and the distinct chemical characteristics of both OPs and oximes documented in numerous X-ray structures of OP-conjugated AChEs. Efforts to improve oxime reactivation efficacy by AChE structure-based enhancement of oxime structure have yielded only limited success. We outline here the potential limitations of available AChE X-ray structures that preclude an accurate prediction of oxime structures, which are necessary for association in the OP-AChE gorge and nucleophilic attack of the OP-conjugated phosphorus.

The PKA regulatory subunit from yeast forms a homotetramer: Low-resolution structure of the N-terminal oligomerization domain.

The cAMP dependent protein kinase (PKA) is a key enzyme involved in many cellular processes in eukaryotes. In mammals, the regulatory (R) subunit localises the catalytic (C) subunit to specific subcellular sites through the interaction of its N-terminal homodimeric docking and dimerization (D/D) domain with specific scaffold proteins. The structure of the D/D domain has been extensively studied in mammals, but there is little information from non-mammalian species. In this work, we present the structural analysis of the D/D domain of Bcy1, the R subunit of PKA from Saccharomyces cerevisiae. Using chemical crosslinking experiments and static light scattering measurements we found that this R subunit forms a tetramer in solution, unlike its dimeric mammalian counterparts. We determined that the D/D domain is responsible for this unusual oligomeric state. Using biophysical techniques including size-exclusion chromatography, sucrose gradient sedimentation, small angle X-ray scattering (SAXS), and circular dichroism, we performed a detailed structural characterization of the tetrameric D/D domain of Bcy1. We used homology modelling in combination with computer-aided docking methods and ab initio SAXS modelling methods to develop structural models for the D/D domain tetramer. The models consist of two homodimers with a canonical D/D domain fold that generate a dimer of dimers with novel putative interaction surfaces. These findings indicate that the oligomerization states of PKA R subunits is more diverse than previously thought, and suggest that this might allow some forms of PKA to interact with a wide range of intracellular partners.

The Development and Validation of the Interprofessional Attitudes Scale: Assessing the Interprofessional Attitudes of Students in the Health Professions.

PURPOSE: No validated tools assess all four competency domains described in the 2011 report Core Competencies for Interprofessional Collaborative Practice (IPEC Report). The purpose of this study was to develop and validate a tool based on the IPEC Report core competency domains that assesses the interprofessional attitudes of students in the health professions. METHOD: In 2012, an interprofessional team of students and two of the authors developed and administered a survey to students from four colleges and schools at the University of Utah Health Sciences Center (Health, Medicine, Nursing, and Pharmacy). The authors randomly split the responses with complete data into two independent subsets: one for exploratory factor analysis (EFA), the other for confirmatory factor analysis (CFA). They performed these analyses to validate the tool, eliminate redundant questions, and identify subscales. Their analyses focused on aligning tool subscales with the IPEC Report core competencies and demonstrating good construct validity and internal consistency reliability. RESULTS: Of 1,549 students invited, 701 (45.3%) responded. The EFA produced a 27-item scale, with five subscales: teamwork, roles, and responsibilities; patient-centeredness; interprofessional biases; diversity and ethics; and community-centeredness (Cronbach alpha coefficients: 0.62 to 0.92). The CFA indicated that the content of the five subscales was consistent with the EFA model. CONCLUSIONS: The Interprofessional Attitudes Scale (IPAS) is a novel tool that, compared with previous assessment instruments, better reflects current thinking about interprofessional competencies. IPAS should prove useful to health sciences institutions committed to training students to work collaboratively in interprofessional teams.

Prevention of Venous Neointimal Hyperplasia by a Multitarget Receptor Tyrosine Kinase Inhibitor.

BACKGROUND/AIMS: Venous neointimal hyperplasia (NH) is the predominant cause of stenosis in hemodialysis arteriovenous grafts (AVG), but there is currently no clinically used therapy to prevent NH. METHODS: A porcine AVG model was used to identify potential pharmacological targets to prevent NH. Sunitinib, a broad-spectrum tyrosine kinase inhibitor, was examined as a potential anti-NH drug utilizing in vitro and ex vivo models. RESULTS: In an in vivo porcine model, PDGF, VEGF and their receptors PDGFR-α and VEGFR-2 were upregulated at the venous anastomosis within 2 weeks after AVG placement, with NH development by 4 weeks. Sunitinib inhibited PDGF-stimulated proliferation, migration, phosphorylation of MAPK and PI3K/Akt proteins and changes in the expression of cell-cycle regulatory proteins in vascular smooth-muscle cells as well as VEGF-stimulated endothelial cell proliferation in vitro. In an ex vivo model, significant NH was observed in porcine vein segments perfused for 12 days under pathological shear stress. Sunitinib (100 nM) inhibited NH formation, with the intima-to-lumen area ratio decreasing from 0.45 ± 0.25 to 0.04 ± 0.02 (p < 0.05) with treatment. CONCLUSION: These findings demonstrate sunitinib to be a potential NH-preventive drug as well as the utility of an ex vivo model to investigate pharmacotherapies under pathophysiological flow conditions.

Aberrant soluble epoxide hydrolase and oxylipin levels in a porcine arteriovenous graft stenosis model.

Synthetic arteriovenous grafts (AVGs) used for hemodialysis frequently fail due to the development of neointimal hyperplasia (NH) at the vein-graft anastomosis. Inflammation and smooth-muscle cell (SMC) and myofibroblast proliferation and migration likely play an important role in the pathogenesis of NH. Epoxyeicosatrienoic acids (EETs), the products of the catabolism of arachidonic acid by cytochrome P450 enzymes, possess anti-inflammatory, antiproliferative, antimigratory and vasodilatory properties that should reduce NH. The degradation of vasculoprotective EETs is catalyzed by the enzyme, soluble epoxide hydrolase (sEH). sEH upregulation may thus contribute to NH development by the enhanced removal of vasculoprotective EETs. In this study, sEH, cytochrome P450 and EETs were examined after AVG placement in a porcine model to explore their potential roles in AVG stenosis. Increased sEH protein expression, decreased P450 epoxygenase activity and dysregulation of 5 oxylipin mediators were observed in the graft-venous anastomotic tissues when compared to control veins. Pharmacological inhibitors of sEH decreased the growth factor-induced migration of SMCs and fibroblasts, although they had no significant effect on the proliferation of these cells. These results provide insights on epoxide biology in vascular disorders and a rationale for the development of novel pharmacotherapeutic strategies to prevent AVG failure due to NH and stenosis.

The roles of the RIIß linker and N-terminal cyclic nucleotide-binding domain in determining the unique structures of the type IIß protein kinase A: a small angle x-ray and neutron scattering study.

Protein kinase A (PKA) is ubiquitously expressed and is responsible for regulating many important cellular functions in response to changes in intracellular cAMP concentrations. The PKA holoenzyme is a tetramer (R2:C2), with a regulatory subunit homodimer (R2) that binds and inhibits two catalytic (C) subunits; binding of cAMP to the regulatory subunit homodimer causes activation of the catalytic subunits. Four different R subunit isoforms exist in mammalian cells, and these confer different structural features, subcellular localization, and biochemical properties upon the PKA holoenzymes they form. The holoenzyme containing RIIß is structurally unique in that the type IIß holoenzyme is much more compact than the free RIIß homodimer. We have used small angle x-ray scattering and small angle neutron scattering to study the solution structure and subunit organization of a holoenzyme containing an RIIß C-terminal deletion mutant (RIIß(1-280)), which is missing the C-terminal cAMP-binding domain to better understand the structural organization of the type IIß holoenzyme and the RIIß domains that contribute to stabilizing the holoenzyme conformation. Our results demonstrate that compaction of the type IIß holoenzyme does not require the C-terminal cAMP-binding domain but rather involves large structural rearrangements within the linker and N-terminal cyclic nucleotide-binding domain of the RIIß homodimer. The structural rearrangements are significantly greater than seen previously with RIIα and are likely to be important in mediating short range and long range interdomain and intersubunit interactions that uniquely regulate the activity of the type IIß isoform of PKA.

PKA RIα homodimer structure reveals an intermolecular interface with implications for cooperative cAMP binding and Carney complex disease.

The regulatory (R) subunit is the cAMP receptor of protein kinase A. Following cAMP binding, the inactive PKA holoenzyme complex separates into two active catalytic (C) subunits and a cAMP-bound R dimer. Thus far, only monomeric R structures have been solved, which fell short in explaining differences of cAMP binding for the full-length protein as compared to the truncated R subunits. Here we solved a full-length R-dimer structure that reflects the biologically relevant conformation, and this structure agrees well with small angle X-ray scattering. An isoform-specific interface is revealed between the protomers. This interface acts as an intermolecular sensor for cAMP and explains the cooperative character of cAMP binding to the RIα dimer. Mutagenesis of residues on this interface not only leads to structural and biochemical changes, but is also linked to Carney complex disease.

A kinase interacting protein (AKIP1) is a key regulator of cardiac stress.

cAMP-dependent protein kinase (PKA) regulates a myriad of functions in the heart, including cardiac contractility, myocardial metabolism,and gene expression. However, a molecular integrator of the PKA response in the heart is unknown. Here, we show that the PKA adaptor A-kinase interacting protein 1 (AKIP1) is up-regulated in cardiac myocytes in response to oxidant stress. Mice with cardiac gene transfer of AKIP1 have enhanced protection to ischemic stress. We hypothesized that this adaptation to stress was mitochondrial dependent. AKIP1 interacted with the mitochondrial localized apoptosis inducing factor (AIF) under both normal and oxidant stress. When cardiac myocytes or whole hearts are exposed to oxidant and ischemic stress, levels of both AKIP1 and AIF were enhanced. AKIP1 is preferentially localized to interfibrillary mitochondria and up-regulated in this cardiac mitochondrial subpopulation on ischemic injury. Mitochondria isolated from AKIP1 gene transferred hearts showed increased mitochondrial localization of AKIP1, decreased reactive oxygen species generation, enhanced calcium tolerance, decreased mitochondrial cytochrome C release,and enhance phosphorylation of mitochondrial PKA substrates on ischemic stress. These observations highlight AKIP1 as a critical molecular regulator and a therapeutic control point for stress adaptation in the heart.