Jeremy W. Chambers

Mitochondrial c-jun-N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.

Atmospheric oxygen inhibits growth and differentiation of marrow-derived mouse mesenchymal stem cells via a p53-dependent mechanism: implications for long-term culture expansion.

Large scale expansion of human mesenchymal stem cells (MSCs) is routinely performed for clinical therapy. In contrast, developing protocols for large scale expansion of primary mouse MSCs has been more difficult due to unique aspects of rodent biology. Currently, established methods to isolate mouse MSCs select for rapidly dividing subpopulations that emerge from bone marrow cultures following long‐term (months) expansion in atmospheric oxygen. Herein, we demonstrate that exposure to atmospheric oxygen rapidly induced p53, TOP2A, and BCL2‐associated X protein (BAX) expression and mitochondrial reactive oxygen species (ROS) generation in primary mouse MSCs resulting in oxidative stress, reduced cell viability, and inhibition of cell proliferation. Alternatively, procurement and culture in 5% oxygen supported more prolific expansion of the CD45−ve/CD44+ve cell fraction in marrow, produced increased MSC yields following immunodepletion, and supported sustained MSC growth resulting in a 2,300‐fold increase in cumulative cell yield by fourth passage. MSCs cultured in 5% oxygen also exhibited enhanced trilineage differentiation. The oxygen‐induced stress response was dependent upon p53 since siRNA‐mediated knockdown of p53 in wild‐type cells or exposure of p53−/− MSCs to atmospheric oxygen failed to induce ROS generation, reduce viability, or arrest cell growth. These data indicate that long‐term culture expansion of mouse MSCs in atmospheric oxygen selects for clones with absent or impaired p53 function, which allows cells to escape oxygen‐induced growth inhibition. In contrast, expansion in 5% oxygen generates large numbers of primary mouse MSCs that retain sensitivity to atmospheric oxygen, and therefore a functional p53 protein, even after long‐term expansion in vitro. STEM CELLS 2012;30:975–987

Synthesis, Biological Evaluation, X-ray Structure, and Pharmacokinetics of Aminopyrimidine c-jun-N-terminal Kinase (JNK) Inhibitors

Given the significant body of data supporting an essential role for c-jun-N-terminal kinase (JNK) in neurodegenerative disorders, we set out to develop highly selective JNK inhibitors with good cell potency and good brain penetration properties. The structure-activity relationships (SAR) around a series of aminopyrimidines were evaluated utilizing biochemical and cell-based assays to measure JNK inhibition and brain penetration in mice. Microsomal stability in three species, P450 inhibition, inhibition of generation of reactive oxygen species (ROS), and pharmacokinetics in rats were also measured. Compounds 9g, 9i, 9j, and 9l had greater than 135-fold selectivity over p38, and cell-based IC(50) values < 100 nM. Moreover, compound 9l showed an IC(50) = 0.8 nM for inhibition of ROS and had good pharmacokinetic properties in rats along with a brain-to-plasma ratio of 0.75. These results suggest that biaryl substituted aminopyrimidines represented by compound 9l may serve as the first small molecule inhibitors to test efficacy of JNK inhibitors in neurodegenerative disorders.

Blocking c-jun-N-terminal Kinase (JNK) Translocation to the Mitochondria Prevents 6-hydroxydopamine-induced Toxicity in vitro and in vivo

Background: Little is known about the role for JNK mitochondrial signaling in neuronal cell death. Results: Global and mitochondrial inhibition of JNK protects against 6-OHDA-induced neuronal loss in the SNpc. Conclusion: Blocking JNK mitochondrial translocation or JNK inhibition may be an effective treatment for neuronal death in Parkinson disease. Significance: These findings suggest a new molecular target for JNK inhibition. Because oxidative stress and mitochondrial dysfunction are well known contributors to Parkinson disease (PD), we set out to investigate the role mitochondrial JNK plays in the etiology of 6-hydroxydopamine-induced (6-OHDA) oxidative stress, mitochondrial dysfunction, and neurotoxicity in SHSY5Y cells and neuroprotection and motor behavioral protection in vivo. To do this, we utilized a cell-permeable peptide of the outer mitochondrial membrane protein, Sab (SH3BP5), as an inhibitor of JNK mitochondrial translocation. In vitro studies showed that 6-OHDA induced JNK translocation to the mitochondria and that inhibition of mitochondrial JNK signaling by Tat-SabKIM1 protected against 6-OHDA-induced oxidative stress, mitochondrial dysfunction, and neurotoxicity. Administration of Tat-SabKIM1 via an intracerebral injection into the mid-forebrain bundle increased the number of tyrosine hydroxylase immunoreactive neurons in the substantia nigra pars compacta by 2-fold (p < 0.05) in animals lesioned with 6-OHDA, compared with animals treated only with 6-OHDA into the nigrostriatal pathway. In addition, Tat-SabKIM1 decreased the d-amphetamine-induced unilateral rotations associated with the lesion by 30% (p < 0.05). Steady-state brain levels of Tat-SabKIM1 at day 7 were 750 nm, which was ∼3.4-fold higher than the IC50 for this peptide versus Sab protein. Collectively, these data suggest that 6-OHDA induced JNK translocation to the mitochondria and that blocking this translocation reduced oxidative stress, mitochondrial dysfunction, and neurotoxicity both in vitro and in vivo. Moreover, the data suggest that inhibitors that block association of JNKs with the mitochondria may be useful neuroprotective agents for the treatment of Parkinson disease.

Inhibition of JNK Mitochondrial Localization and Signaling Is Protective against Ischemia/Reperfusion Injury in Rats

Background: Little is known about the role of JNK mitochondrial signaling in cardiomyocyte cell death. Results: Global and mitochondrial inhibition of JNK protects against I/R injury thus reducing infarct volume. Conclusion: Blocking JNK mitochondrial translocation or JNK inhibition may be an effective treatment for I/R-induced cardiomyocyte death. Significance: These findings suggest a new molecular target for JNK inhibition. To build upon recent findings that mitochondrial JNK signaling is inhibited by selectively blocking the interaction between JNK and Sab, we utilized a cell-permeable peptide to demonstrate that ischemia/reperfusion (I/R) injury could be protected in vivo and that JNK mitochondrial signaling was the mechanism by which reactive oxygen species (ROS) generation, mitochondrial dysfunction, and cardiomyocyte cell death occur. We also demonstrated that 5 mg/kg SR-3306 (a selective JNK inhibitor) was able to protect against I/R injury, reducing infarct volume by 34% (p < 0.05) while also decreasing I/R-induced increases in the activity of creatine phosphokinase and creatine kinase-MB. TUNEL staining showed that the percent TUNEL positive nuclei in rat hearts increased 10-fold after I/R injury and that this was reduced 4-fold (p < 0.01) by SR-3306. These data suggest that blocking JNK mitochondrial translocation or JNK inhibition prevents ROS increases and mitochondrial dysfunction and may be an effective treatment for I/R-induced cardiomyocyte death.

Assembly of heterohexameric trypanosome hexokinases reveals that hexokinase 2 is a regulable enzyme.

Glycolysis is essential to Trypanosoma brucei, the protozoan parasite that causes African sleeping sickness in humans and nagana in cattle. Hexokinase (HK), the first enzyme in glycolysis, catalyzes the phosphorylation of glucose to form glucose 6-phosphate. T. brucei harbors two HKs that are 98% identical at the amino acid level, T. brucei hexokinase 1 (TbHK1) and TbHK2. Recombinant TbHK1 (rTbHK1) has HK activity, whereas rTbHK2 does not. Unlike other eukaryotic HKs, TbHK1 is not subject to inhibition by ADP and glucose 6-phosphate. However, TbHK1 is inhibited by myristate, a critical fatty acid in T. brucei biology. We report here that rTbHKs, similar to authentic TbHK, form oligomers. Myristate dissociated these assemblies when incubated with either ATP or glucose. Furthermore, oligomer disruption was reversible by removal of myristate. Mixing of rTbHK1 and rTbHK2 monomers followed by reassembly yielded enzyme with an ∼3-fold increase in specific activity compared with similarly treated rTbHK1 alone. Surprisingly, reassembly of rTbHK2 with an inactive rTbHK1 variant yielded an active HK, revealing for the first time that rTbHK2 is competent for HK activity. Finally, pyrophosphate inhibits active reassembled rTbHK2 oligomers but not oligomeric rTbHK1, suggesting that the two enzymes have distinct regulatory mechanisms.

Activity of a Second Trypanosoma brucei Hexokinase Is Controlled by an 18-Amino-Acid C-Terminal Tail

ABSTRACT Trypanosoma brucei expresses two hexokinases that are 98% identical, namely, TbHK1 and TbHK2. Homozygous null TbHK2−/− procyclic-form parasites exhibit an increased doubling time, a change in cell morphology, and, surprisingly, a twofold increase in cellular hexokinase activity. Recombinant TbHK1 enzymatic activity is similar to that of other hexokinases, with apparent Km values for glucose and ATP of 0.09 ± 0.02 mM and 0.28 ± 0.1 mM, respectively. The kcat value for TbHK1 is 2.9 × 104 min−1. TbHK1 can use mannose, fructose, 2-deoxyglucose, and glucosamine as substrates. In addition, TbHK1 is inhibited by fatty acids, with lauric, myristic, and palmitic acids being the most potent (with 50% inhibitory concentrations of 75.8, 78.4, and 62.4 μM, respectively). In contrast to TbHK1, recombinant TbHK2 lacks detectable enzymatic activity. Seven of the 10 amino acid differences between TbHK1 and TbHK2 lie within the C-terminal 18 amino acids of the polypeptides. Modeling of the proteins maps the C-terminal tails near the interdomain cleft of the enzyme that participates in the conformational change of the enzyme upon substrate binding. Replacing the last 18 amino acids of TbHK2 with the corresponding residues of TbHK1 yields an active recombinant protein with kinetic properties similar to those of TbHK1. Conversely, replacing the C-terminal tail of TbHK1 with the TbHK2 tail inactivates the enzyme. These findings suggest that the C-terminal tail of TbHK1 is important for hexokinase activity. The altered C-terminal tail of TbHK2, along with the phenotype of the knockout parasites, suggests a distinct function for the protein.

Selective inhibition of mitochondrial JNK signaling achieved using peptide mimicry of the Sab kinase interacting motif-1 (KIM1).

The c-jun N-terminal kinases (JNKs) are responsive to stress stimuli leading to activation of proapoptotic proteins and transcription. Additionally, JNK mitochondrial localization has been reported. To selectively target mitochondrial JNK signaling, we exploited JNK interaction with its mitochondrial scaffold, Sab, using small interfering RNAs (siRNAs) and a cell-permeable peptide corresponding to the KIM1 domain of Sab. Gene silencing and peptide interference of this interaction disrupted JNK translocation to the mitochondria and reduced phosphorylation of Bcl-2 without significant impact on c-Jun phosphorylation or AP-1 transcription. In contrast, the JNK inhibitory peptide (TI-JIP1) prevented these three functions. Tat-Sab(KIM1) selectivity was also demonstrated in anisomycin-stressed HeLa cells where Tat-Sab(KIM1) prevented Bcl-2 phosphorylation, cell death, loss of mitochondrial membrane potential, and superoxide generation but not c-Jun phosphorylation. Conversely, TI-JIP1 prevented all aforementioned stress-induced events. This probe introduces a means to evaluate JNK-mediated events on the mitochondria without intervening in nuclear functions of JNK.

A Small Molecule Bidentate-Binding Dual Inhibitor Probe of the LRRK2 and JNK Kinases

Both JNK and LRRK2 are associated with Parkinsons disease (PD). Here we report a reasonably selective and potent kinase inhibitor (compound 6) that bound to both JNK and LRRK2 (a dual inhibitor). A bidentate-binding strategy that simultaneously utilized the ATP hinge binding and a unique protein surface site outside of the ATP pocket was applied to the design and identification of this kind of inhibitor. Compound 6 was a potent JNK3 and modest LRRK2 dual inhibitor with an enzyme IC50 value of 12 nM and 99 nM (LRRK2-G2019S), respectively. Compound 6 also exhibited good cell potency, inhibited LRRK2:G2019S-induced mitochondrial dysfunction in SHSY5Y cells, and was demonstrated to be reasonably selective against a panel of 116 kinases from representative kinase families. Design of such a probe molecule may help enable testing if dual JNK and LRRK2 inhibitions have added or synergistic efficacy in protecting against neurodegeneration in PD.

A rapid and sensitive high-throughput screening method to identify compounds targeting protein–nucleic acids interactions

DNA-binding and RNA-binding proteins are usually considered ‘undruggable’ partly due to the lack of an efficient method to identify inhibitors from existing small molecule repositories. Here we report a rapid and sensitive high-throughput screening approach to identify compounds targeting protein–nucleic acids interactions based on protein–DNA or protein–RNA interaction enzyme-linked immunosorbent assays (PDI-ELISA or PRI-ELISA). We validated the PDI-ELISA method using the mammalian high-mobility-group protein AT-hook 2 (HMGA2) as the protein of interest and netropsin as the inhibitor of HMGA2–DNA interactions. With this method we successfully identified several inhibitors and an activator for HMGA2–DNA interactions from a collection of 29 DNA-binding compounds. Guided by this screening excise, we showed that netropsin, the specific inhibitor of HMGA2–DNA interactions, strongly inhibited the differentiation of the mouse pre-adipocyte 3T3-L1 cells into adipocytes, most likely through a mechanism by which the inhibition is through preventing the binding of HMGA2 to the target DNA sequences. This method should be broadly applicable to identify compounds or proteins modulating many DNA-binding or RNA-binding proteins.