Thermal Acclimation of the Symbiotic Alga Symbiodinium spp. Alleviates Photobleaching under Heat Stress

A moderate increase in seawater temperature causes coral bleaching, at least partially through photobleaching of the symbiotic algae Symbiodinium spp. Photobleaching of Symbiodinium spp. is primarily associated with the loss of light-harvesting proteins of photosystem II (PSII) and follows the inactivation of PSII under heat stress. Here, we examined the effect of increased growth temperature on the change in sensitivity of Symbiodinium spp. PSII inactivation and photobleaching under heat stress. When Symbiodinium spp. cells were grown at 25°C and 30°C, the thermal tolerance of PSII, measured by the thermal stability of the maximum quantum yield of PSII in darkness, was commonly enhanced in all six Symbiodinium spp. tested. In Symbiodinium sp. CCMP827, it took 6 h to acquire the maximum PSII thermal tolerance after transfer from 25°C to 30°C. The effect of increased growth temperature on the thermal tolerance of PSII was completely abolished by chloramphenicol, indicating that the acclimation mechanism of PSII is associated with the de novo synthesis of proteins. When CCMP827 cells were exposed to light at temperature ranging from 25°C to 35°C, the sensitivity of cells to both high temperature-induced photoinhibition and photobleaching was ameliorated by increased growth temperatures. These results demonstrate that thermal acclimation of Symbiodinium spp. helps to improve the thermal tolerance of PSII, resulting in reduced inactivation of PSII and algal photobleaching. These results suggest that whole-organism coral bleaching associated with algal photobleaching can be at least partially suppressed by the thermal acclimation of Symbiodinium spp. at higher growth temperatures.Reef-building corals harbor symbiotic dinoflagellate algae of the genus Symbiodinium. Corals generally show a brownish coloration due to algal photosynthetic pigments, such as peridinin and chlorophylls a and c₂ present in in situ Symbiodinium spp. However, under increased seawater temperatures, corals become pale through the loss of Symbiodinium spp. cells and/or the loss of photosynthetic pigments of in situ Symbiodinium spp. (Glynn, 1993, 1996; Hoeghguldberg, 1999; Fitt et al., 2001; Coles and Brown, 2003). This phenomenon is so-called coral bleaching. Since a healthy algae-coral symbiotic relationship is important for coral survival (Yellowlees et al., 2008), severe coral bleaching leads to the mortality of corals and even the destruction of entire coral reef ecosystems. The frequency and intensity of coral bleaching have been increasing since the early 1980s, and it is predicted to become more severe in the future due to ongoing global climate change and warming (Hughes et al., 2003). Coral reef ecosystems are in serious decline, with an estimated 30% already severely damaged, and it is predicted that globally as much as 60% of the world’s coral reef ecosystems may be lost by 2030 (Hughes et al., 2003).Coral bleaching caused by heat stress is at least partially attributed to the photobleaching of photosynthetic pigments in Symbiodinium spp. within corals (Kleppel et al., 1989; Porter et al., 1989; Fitt et al., 2001; Takahashi et al., 2004; Venn et al., 2006). The photobleaching commonly occurs in photosynthetic organisms under conditions where the absorbed light energy for photosynthesis is in excess of the capacity to use it, particularly under environmental stress conditions in high light (Niyogi, 1999). In cultured Symbiodinium spp. cells, heat stress-associated algal photobleaching is attributed to the loss of major light-harvesting proteins, such as the peridinin-chlorophyll a-binding proteins and the chlorophyll a-chlorophyll c₂-peridin protein complexes (Takahashi et al., 2008). A recent study has also demonstrated that the heat stress-associated loss of light-harvesting proteins in Symbiodinium spp. is attributed to suppression of the de novo synthesis of light-harvesting proteins but not acceleration of the photodamage and subsequent degradation of light-harvesting proteins (Takahashi et al., 2008). High-temperature sensitivity of Symbiodinium spp. cells to photobleaching differs among Symbiodinium spp., and this is at least partially attributed to the thermal sensitivity of the de novo synthesis of light-harvesting proteins (Takahashi et al., 2008).Heat stress-associated photobleaching in Symbiodinium spp. follows severe photoinhibition of PSII (Takahashi et al., 2008). The extent of photoinhibition is a result of the dynamic balance between the rate of photodamage to PSII and the rate of its repair. In plants and green algae, the PSII repair process is primarily composed of the degradation and the de novo synthesis of the D1 proteins in photodamaged PSII protein complexes (Aro et al., 1993; Takahashi and Murata, 2008; Takahashi and Badger, 2011). However, this differs in Symbiodinium spp., in that the photodamaged PSII can be repaired without the de novo synthesis of D1 proteins (Takahashi et al., 2009b). Furthermore, a part of photodamaged PSII is repaired without protein synthesis (Takahashi et al., 2009b), indicating that Symbiodinium spp. have a unique PSII repair mechanism. In Symbiodinium spp. found within corals and also in culture, heat stress accelerates photoinhibition at least partially through the suppression of PSII repair (Warner et al., 1999; Takahashi et al., 2004, 2009b). However, the sensitivity of PSII repair to heat stress differs among Symbiodinium spp. and is strongly related to the sensitivity of PSII to photoinhibition under heat stress (Takahashi et al., 2009b).The high-temperature sensitivity of corals to bleaching is changed by their growth temperature, and this is suggested to be due to changing in situ Symbiodinium spp. populations from heat-sensitive to heat-resistant ecotypes (Baker, 2001, 2003; Baker et al., 2004; Berkelmans and van Oppen, 2006; Jones et al., 2008; Jones and Berkelmans, 2010). However, thermal tolerance of the population might also be enhanced by thermal acclimation mechanism(s) associated with both the corals and Symbiodinium spp., although experimental data that directly support this hypothesis are lacking. In this study, we examine the effect of increased growth temperature (thermal acclimation treatment) on the extent of heat stress-associated algal photobleaching using cultured Symbiodinium spp. Our results demonstrate that Symbiodinium spp. commonly have thermal acclimation mechanisms that enhance the high-temperature tolerance of PSII and alleviate heat stress-associated photobleaching. Our results strongly suggest that thermal acclimation of Symbiodinium spp. plays a role in alleviating algal photobleaching-associated coral bleaching under heat stress.     

Depletion of white adipocyte progenitors induces beige adipocyte differentiation and suppresses obesity development

Overgrowth of white adipose tissue (WAT) in obesity occurs as a result of adipocyte hypertrophy and hyperplasia. Expansion and renewal of adipocytes relies on proliferation and differentiation of white adipocyte progenitors (WAP); however, the requirement of WAP for obesity development has not been proven. Here, we investigate whether depletion of WAP can be used to prevent WAT expansion. We test this approach by using a hunter-killer peptide designed to induce apoptosis selectively in WAP. We show that targeted WAP cytoablation results in a long-term WAT growth suppression despite increased caloric intake in a mouse diet-induced obesity model. Our data indicate that WAP depletion results in a compensatory population of adipose tissue with beige adipocytes. Consistent with reported thermogenic capacity of beige adipose tissue, WAP-depleted mice display increased energy expenditure. We conclude that targeting of white adipocyte progenitors could be developed as a strategy to sustained modulation of WAT metabolic activity.Obesity, a medical condition predisposing to diabetes, cardiovascular diseases, cancer, and complicating other life-threatening diseases, is becoming an increasingly important social problem.^{1, 2, 3} Development of pharmacological approaches to reduction of body fat has remained a daunting task.⁴ Approved obesity treatments typically produce only moderate and temporary effects.^2,5 White adipocytes are the differentiated cells of white adipose tissue (WAT) that store triglycerides in lipid droplets.^6,7 In contrast, adipocytes of brown adipose tissue (BAT) dissipate excess energy through adaptive thermogenesis. Under certain conditions, white adipocytes can become partially replaced with brown-like ‘beige'' (‘brite'') adipocytes that simulate the thermogenic function of BAT adipocytes.^7,8 Obesity develops in the context of positive energy balance as a result of hypertrophy and hyperplasia of white adipocytes.⁹Expansion and renewal of the white adipocyte pool in WAT continues in adulthood.^10,11 This process is believed to rely on proliferation and self-renewal of mesenchymal precursor cells¹² that we term white adipocyte progenitors (WAPs). WAPs reside within the population of adipose stromal cells (ASCs)¹³ and are functionally similar to bone marrow mesenchymal stem cells (MSCs).^{14, 15, 16} ASCs can be isolated from the stromal/vascular fraction (SVF) of WAT based on negativity for hematopoietic (CD45) and endothelial (CD31) markers.^17,18 ASCs support vascularization as mural/adventitial cells secreting angiogenic factors^5,19 and, unlike bone marrow MSCs, express CD34.^19,20 WAPs have been identified within the ASC population based on expression of mesenchymal markers, such as platelet-derived growth factor receptor-β (PDGFRβ, aka CD140b) and pericyte markers.^17,18 Recently, a distinct ASC progenitor population capable of differentiating into both white and brown adipocytes has been identified in WAT based on PDGFRα (CD140a) expression and lack of PDGFRβ expression.^21,22 The physiological relevance of the two precursor populations residing in WAT has not been explored.We have previously established an approach to isolate peptide ligands binding to receptors selectively expressed on the surface of cell populations of interest.^{23, 24, 25, 26, 27} Such cell-targeted peptides can be used for targeted delivery of experimental therapeutic agents in vivo. A number of ‘hunter-killer'' peptides²⁸ composed of a cell-homing domain binding to a surface marker and of KLAKLAK₂ (sequence KLAKLAKKLAKLAK), a moiety inducing apoptosis upon receptor-mediated internalization, has been described by our group.^26,29 Such bimodal peptides have been used for depletion of malignant cells and organ-specific endothelial cells in preclinical animal models.^26,30,31 Recently, we isolated a cyclic peptide WAT7 (amino acid sequence CSWKYWFGEC) based on its specific binding to ASCs.²⁰ We identified Δ-decorin (ΔDCN), a proteolytic cleavage fragment of decorin, as the WAT7 receptor specifically expressed on the surface of CD34+PDGFRβ+CD31-CD45- WAPs and absent on MSCs in other organs.²⁰Here, we investigated whether WAPs are required for obesity development in adulthood. By designing a new hunter-killer peptide that directs KLAKLAK₂ to WAPs through WAT7/ΔDCN interaction, we depleted WAP in the mouse diet-induced obesity model. We demonstrate that WAP depletion suppresses WAT growth. We show that, in response to WAP deficiency, WAT becomes populated with beige adipocytes. Consistent with the reported thermogenic function of beige adipocytes,^32,33 the observed WAT remodeling is associated with increased energy expenditure. We identify a population of PDGFRα-positive, PDGFRβ-negative ASCs reported recently²² as a population surviving WAP depletion and responsible for WAT browning.     

The nuclease activity of DNA2 promotes exonuclease 1–independent mismatch repair

The DNA mismatch repair (MMR) system is a major DNA repair system that corrects DNA replication errors. In eukaryotes, the MMR system functions via mechanisms both dependent on and independent of exonuclease 1 (EXO1), an enzyme that has multiple roles in DNA metabolism. Although the mechanism of EXO1-dependent MMR is well understood, less is known about EXO1-independent MMR. Here, we provide genetic and biochemical evidence that the DNA2 nuclease/helicase has a role in EXO1-independent MMR. Biochemical reactions reconstituted with purified human proteins demonstrated that the nuclease activity of DNA2 promotes an EXO1-independent MMR reaction via a mismatch excision-independent mechanism that involves DNA polymerase δ. We show that DNA polymerase ε is not able to replace DNA polymerase δ in the DNA2-promoted MMR reaction. Unlike its nuclease activity, the helicase activity of DNA2 is dispensable for the ability of the protein to enhance the MMR reaction. Further examination established that DNA2 acts in the EXO1-independent MMR reaction by increasing the strand-displacement activity of DNA polymerase δ. These data reveal a mechanism for EXO1-independent mismatch repair.

The mismatch repair (MMR) system has been conserved from bacteria to humans (1, 2). It promotes genome stability by suppressing spontaneous and DNA damage-induced mutations (1, 3, 4, 5, 6, 7, 8, 9, 10, 11). The key function of the MMR system is the correction of DNA replication errors that escape the proofreading activities of replicative DNA polymerases (1, 4, 5, 6, 7, 8, 9, 10, 12). In addition, the MMR system removes mismatches formed during strand exchange in homologous recombination, suppresses homeologous recombination, initiates apoptosis in response to irreparable DNA damage caused by several anticancer drugs, and contributes to instability of triplet repeats and alternative DNA structures (1, 4, 5, 7, 8, 9, 10, 11, 13, 14, 15, 16, 17, 18). The principal components of the eukaryotic MMR system are MutSα (MSH2-MSH6 heterodimer), MutLα (MLH1-PMS2 heterodimer in humans and Mlh1-Pms1 heterodimer in yeast), MutSβ (MSH2-MSH3 heterodimer), proliferating cell nuclear antigen (PCNA), replication factor C (RFC), exonuclease 1 (EXO1), RPA, and DNA polymerase δ (Pol δ). Loss-of-function mutations in the MSH2, MLH1, MSH6, and PMS2 genes of the human MMR system cause Lynch and Turcot syndromes, and hypermethylation of the MLH1 promoter is responsible for ∼15% of sporadic cancers in several organs (19, 20). MMR deficiency leads to cancer initiation and progression via a multistage process that involves the inactivation of tumor suppressor genes and action of oncogenes (21).MMR occurs behind the replication fork (22, 23) and is a major determinant of the replication fidelity (24). The correction of DNA replication errors by the MMR system increases the replication fidelity by ∼100 fold (25). Strand breaks in leading and lagging strands as well as ribonucleotides in leading strands serve as signals that direct the eukaryotic MMR system to remove DNA replication errors (26, 27, 28, 29, 30). MMR is more efficient on the lagging than the leading strand (31). The substrates for MMR are all six base–base mismatches and 1 to 13-nt insertion/deletion loops (25, 32, 33, 34). Eukaryotic MMR commences with recognition of the mismatch by MutSα or MutSβ (32, 34, 35, 36). MutSα is the primary mismatch-recognition factor that recognizes both base–base mismatches and small insertion/deletion loops whereas MutSβ recognizes small insertion/deletion loops (32, 34, 35, 36, 37). After recognizing the mismatch, MutSα or MutSβ cooperates with RFC-loaded PCNA to activate MutLα endonuclease (38, 39, 40, 41, 42, 43). The activated MutLα endonuclease incises the discontinuous daughter strand 5′ and 3′ to the mismatch. A 5'' strand break formed by MutLα endonuclease is utilized by EXO1 to enter the DNA and excise a discontinuous strand portion encompassing the mismatch in a 5''→3′ excision reaction stimulated by MutSα/MutSβ (38, 44, 45). The generated gap is filled in by the Pol δ holoenzyme, and the nick is ligated by a DNA ligase (44, 46, 47). DNA polymerase ε (Pol ε) can substitute for Pol δ in the EXO1-dependent MMR reaction, but its activity in this reaction is much lower than that of Pol δ (48). Although MutLα endonuclease is essential for MMR in vivo, 5′ nick-dependent MMR reactions reconstituted in the presence of EXO1 are MutLα-independent (44, 47, 49).EXO1 deficiency in humans does not seem to cause significant cancer predisposition (19). Nevertheless, it is known that Exo1^-/- mice are susceptible to the development of lymphomas (50). Genetic studies in yeast and mice demonstrated that EXO1 inactivation causes only a modest defect in MMR (50, 51, 52, 53). In agreement with these genetic studies, a defined human EXO1-independent MMR reaction that depends on the strand-displacement DNA synthesis activity of Pol δ holoenzyme to remove the mismatch was reconstituted (54). Furthermore, an EXO1-independent MMR reaction that occurred in a mammalian cell extract system without the formation of a gapped excision intermediate was observed (54). Together, these findings implicated the strand-displacement activity of Pol δ holoenzyme in EXO1-independent MMR.In this study, we investigated DNA2 in the context of MMR. DNA2 is an essential multifunctional protein that has nuclease, ATPase, and 5''→3′ helicase activities (55, 56, 57). Previous research ascertained that DNA2 removes long flaps during Okazaki fragment maturation (58, 59, 60), participates in the resection step of double-strand break repair (61, 62, 63), initiates the replication checkpoint (64), and suppresses the expansions of GAA repeats (65). We have found in vivo and in vitro evidence that DNA2 promotes EXO1-independent MMR. Our data have indicated that the nuclease activity of DNA2 enhances the strand-displacement activity of Pol δ holoenzyme in an EXO1-independent MMR reaction.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Bak apoptotic pores involve a flexible C-terminal region and juxtaposition of the C-terminal transmembrane domains

Bak and Bax mediate apoptotic cell death by oligomerizing and forming a pore in the mitochondrial outer membrane. Both proteins anchor to the outer membrane via a C-terminal transmembrane domain, although its topology within the apoptotic pore is not known. Cysteine-scanning mutagenesis and hydrophilic labeling confirmed that in healthy mitochondria the Bak α9 segment traverses the outer membrane, with 11 central residues shielded from labeling. After pore formation those residues remained shielded, indicating that α9 does not line a pore. Bak (and Bax) activation allowed linkage of α9 to neighboring α9 segments, identifying an α9:α9 interface in Bak (and Bax) oligomers. Although the linkage pattern along α9 indicated a preferred packing surface, there was no evidence of a dimerization motif. Rather, the interface was invoked in part by Bak conformation change and in part by BH3:groove dimerization. The α9:α9 interaction may constitute a secondary interface in Bak oligomers, as it could link BH3:groove dimers to high-order oligomers. Moreover, as high-order oligomers were generated when α9:α9 linkage in the membrane was combined with α6:α6 linkage on the membrane surface, the α6-α9 region in oligomerized Bak is flexible. These findings provide the first view of Bak carboxy terminus (C terminus) membrane topology within the apoptotic pore.Mitochondrial permeabilization during apoptosis is regulated by the Bcl-2 family of proteins.^{1, 2, 3} Although the Bcl-2 homology 3 (BH3)-only members such as Bid and Bim trigger apoptosis by binding to other family members, the prosurvival members block apoptosis by sequestering their pro-apoptotic relatives. Two remaining members, Bak and Bax, form the apoptotic pore within the mitochondrial outer membrane (MOM).Bak and Bax are globular proteins comprising nine α-helices.^{4, 5} They are activated by BH3-only proteins binding to the α2–α5 surface groove,^{6, 7, 8, 9, 10, 11, 12} or for Bax, to the α1/α6 ‘rear pocket''.¹³ Binding triggers dissociation of the latch domain (α6–α8) from the core domain (α2–α5), together with exposure of N-terminal epitopes and the BH3 domain.^{6, 7, 14, 15, 16} The exposed BH3 domain then binds to the hydrophobic groove in another Bak or Bax molecule to generate symmetric homodimers.^{6, 7, 14, 17, 18} In addition to dimerizing, parts of activated Bak and Bax associate with the lipid bilayer.¹⁹ In Bax, the α5 and α6 helices may insert into the MOM,²⁰ although recent studies indicate that they lie in-plane on the membrane surface, with the hydrophobic α5 sandwiched between the membrane and a BH3:groove dimer interface.^{7, 21, 22, 23} The dimers can be linked via cysteine residues placed in α6,^{18, 24, 25} and more recently via cysteine residues in either α3 or α5,^{6, 21} allowing detection of the higher-order oligomers associated with pore formation.^{26, 27} However, whether these interactions are required for high-order oligomers and pore formation remains unclear.Like most Bcl-2 members, Bak and Bax are targeted to the MOM via a hydrophobic C-terminal region. The C terminus targets Bak to the MOM in healthy cells,²⁸ whereas the Bax C terminus is either exposed²⁹ or sequestered within the hydrophobic groove until apoptotic signals trigger Bax translocation.^{5, 30, 31} The hydrophobic stretch is important, as substituting polar or charged residues decreased targeting of Bak and Bax.^{10, 32} Mitochondrial targeting is also controlled by basic residues at the far C termini,^{32, 33, 34} and by interaction with VDAC2^{35, 36} via the Bak and Bax C termini.^{37, 38} Retrotranslocation of Bak and Bax was also altered by swapping the C termini.³⁹The membrane topology of the Bak and Bax C termini before and after apoptosis has not been examined directly, due in part to difficulty in reconstituting oligomers of full-length Bak in artificial membranes. Nor is it known whether the C termini contribute to pore formation by promoting oligomerization or disturbing the membrane. To address these questions synthetic peptides based on the Bak and Bax C termini have been studied in model membranes. The peptides adopt a predominantly α-helical secondary structure,^{40, 41, 42, 43} with orientation affected by lipid composition.^{42, 44, 45} The peptides could also permeabilize lipid vesicles,^{41, 43, 46, 47} suggesting that the C termini in full-length Bak and Bax may contribute to pore formation.Here we examined the membrane topology of the C termini within full-length Bak and Bax in the MOM, both before and after apoptotic pore formation. After pore formation the α9 helices of Bak (and of Bax) became juxtaposed but did not line the surface of a pore. The α9:α9 interaction occurred after Bak activation and conformation change, but was promoted by formation of BH3:groove dimers. Combining linkage at more than one interface indicated that the Bak α9:α9 interface can link BH3:groove dimers to high-order oligomers, and moreover, that the α6–α9 region is flexible in oligomerized Bak.     

Genetic deficiency of neuronal RAGE protects against AGE-induced synaptic injury

Synaptic dysfunction and degeneration is an early pathological feature of aging and age-related diseases, including Alzheimer''s disease (AD). Aging is associated with increased generation and deposition of advanced glycation endproducts (AGEs), resulting from nonenzymatic glycation (or oxidation) proteins and lipids. AGE formation is accelerated in diabetes and AD-affected brain, contributing to cellular perturbation. The extent of AGEs'' involvement, if at all, in alterations in synaptic structure and function is currently unknown. Here we analyze the contribution of neuronal receptor of AGEs (RAGE) signaling to AGE-mediated synaptic injury using novel transgenic neuronal RAGE knockout mice specifically targeted to the forebrain and transgenic mice expressing neuronal dominant-negative RAGE (DN-RAGE). Addition of AGEs to brain slices impaired hippocampal long-term potentiation (LTP). Similarly, treatment of hippocampal neurons with AGEs significantly decreases synaptic density. Such detrimental effects are largely reversed by genetic RAGE depletion. Notably, brain slices from mice with neuronal RAGE deficiency or DN-RAGE are resistant to AGE-induced LTP deficit. Further, RAGE deficiency or DN-RAGE blocks AGE-induced activation of p38 signaling. Taken together, these data show that neuronal RAGE functions as a signal transducer for AGE-induced synaptic dysfunction, thereby providing new insights into a mechanism by which the AGEs–RAGE-dependent signaling cascade contributes to synaptic injury via the p38 MAP kinase signal transduction pathway. Thus, RAGE blockade may be a target for development of interventions aimed at preventing the progression of cognitive decline in aging and age-related neurodegenerative diseases.Advanced glycation endproducts (AGEs) are members of a heterogeneous class of molecules, which modify cellular function by distinct mechanisms, including ligation and activation of signal transduction receptors. The products of non-enzymatic glycation (or oxidation) of proteins and lipids, AGEs contribute to the normal aging process and when accelerated have a causative role in the vasculature complications of diabetes mellitus and several neurodegenerative diseases, including Alzheimer''s (AD), Parkinson''s, and Huntington''s diseases.^{1, 2, 3, 4, 5} In diabetic patients, the concentration of circulating AGEs (serum AGE level) has been reported at 7.2–22 mU/ml (equivalent to 30–88 μg/ml AGE-BSA), which is significantly higher than that of non-diabetic patients (3 mU/ml, equivalent to 12 μg/ml AGE-BSA).^{6, 7, 8} The brain AGE level was also increased to 5-6 μM (equivalent to 325–390 μg/ml AGE-BSA) in the diabetic animal model.⁹ Excess AGE accumulation is detrimental to neurons and is believed to be a key to the pathogenesis of cognitive decline in normal aging and specific chronic diseases of aging. For example, in a recent clinical study, peripheral AGE levels were associated with cognitive decline in older adults with and without diabetes.¹⁰ Diabetes complications affect the brain, increasing risk for depression, dementia, and AD. In fact, patients with type 2 diabetes are at twofold to threefold increased relative risk for AD^{11, 12, 13, 14, 15, 16, 17, 18} and accelerated cognitive dysfunction.Long-lived proteins such as β-amyloid peptide (Aβ) and hyperphosphorylated tau protein that accumulate in AD brain are highly susceptible to AGE modification.^{19, 20, 21, 22} AGE-modified Aβ or tau protein results in increased oxidative stress and chronic inflammation, accelerating AD pathology and neuronal perturbation.^{19, 20, 22, 23, 24, 25} Moreover, Aβ or tau glycation results in increased aggregation and subsequent formation of senile plaques or neurofibrillary tangles, the major pathological feature of AD,^{19, 22} suggesting that AGE modification is an important risk factor for neurodegenerative diseases.²⁶ Although increased accumulation of AGEs in brain, as seen in aging, diabetes, or neurodegenerative diseases, speeds up oxidative damage to neurons contributing to synaptic dysfunction and cognitive decline, its underlying mechanisms are not well understood.Receptor for advanced glycation endproduct (RAGE) was first identified as a cell surface receptor of the immunoglobulin superfamily for AGEs.^{27, 28} Increased expression of RAGE occurs in neuronal and non-neuronal cells in the peripheral and central nervous system in aging, diabetes, and AD-affected individuals, where RAGE ligands are upregulated.^{29, 30} Although it has been shown that AGEs–RAGE interaction contributes to cellular perturbation relevant to the pathogenesis of the cardiovascular disease and the diabetes vascular complications,^{31, 32, 33} little is known about the role of AGEs and its interaction with RAGE on synaptic dysfunction. To understand the mechanisms involved in AGE-mediated synaptic damage, the following questions need to be addressed: (1) ‘Do AGEs alter synaptic structure and function? If so, are these changes dependent on RAGE signaling?'' (2) ‘Does RAGE blockage by genetic depletion protect from AGE-induced synaptic dysfunction and loss?'' and (3) ‘What is the impact of neuronal RAGE in AGE-induced aberrant synaptic function?''. Thus it is important to evaluate the impact of AGEs–RAGE interaction on synaptic dysfunction and to explore the mechanism underlying AGE–RAGE-dependent signal transduction and its contribution to synaptic damage.Here we investigate neuronal RAGE signaling in AGE-induced synaptic injury using our novel conditional RAGE knockout mice targeted to cortical neurons as well as transgenic mice that overexpress signal transduction-deficient mutants of RAGE in neurons. Given that neuronal and non-neuronal cells in the brain may contribute to AGE-induced sustained neuronal and synaptic stress and dysfunction, we assessed the impact of global RAGE deletion in this setting and further delineated the mechanism by which RAGE-dependent activation of p38 MAP kinase potentiates AGE-insulted synaptic injury.     

Autophagy is a regulator of TGF-β1-induced fibrogenesis in primary human atrial myofibroblasts

Transforming growth factor-β₁ (TGF-β₁) is an important regulator of fibrogenesis in heart disease. In many other cellular systems, TGF-β₁ may also induce autophagy, but a link between its fibrogenic and autophagic effects is unknown. Thus we tested whether or not TGF-β₁-induced autophagy has a regulatory function on fibrosis in human atrial myofibroblasts (hATMyofbs). Primary hATMyofbs were treated with TGF-β₁ to assess for fibrogenic and autophagic responses. Using immunoblotting, immunofluorescence and transmission electron microscopic analyses, we found that TGF-β₁ promoted collagen type Iα2 and fibronectin synthesis in hATMyofbs and that this was paralleled by an increase in autophagic activation in these cells. Pharmacological inhibition of autophagy by bafilomycin-A1 and 3-methyladenine decreased the fibrotic response in hATMyofb cells. ATG7 knockdown in hATMyofbs and ATG5 knockout (mouse embryonic fibroblast) fibroblasts decreased the fibrotic effect of TGF-β₁ in experimental versus control cells. Furthermore, using a coronary artery ligation model of myocardial infarction in rats, we observed increases in the levels of protein markers of fibrosis, autophagy and Smad2 phosphorylation in whole scar tissue lysates. Immunohistochemistry for LC3β indicated the localization of punctate LC3β with vimentin (a mesenchymal-derived cell marker), ED-A fibronectin and phosphorylated Smad2. These results support the hypothesis that TGF-β₁-induced autophagy is required for the fibrogenic response in hATMyofbs.Interstitial fibrosis is common to many cardiovascular disease etiologies including myocardial infarction (MI),¹ diabetic cardiomyopathy² and hypertension.³ Fibrosis may arise due to maladaptive cardiac remodeling following injury and is a complex process resulting from activation of signaling pathways, such as TGF-β₁.⁴ TGF-β₁ signaling has broad-ranging effects that may affect cell growth, differentiation and the production of extracellular matrix (ECM) proteins.^{5, 6} Elevated TGF-β₁ is observed in post-MI rat heart⁷ and is associated with fibroblast-to-myofibroblast phenoconversion and concomitant activation of canonical Smad signaling.⁸ The result is a proliferation of myofibroblasts, which then leads to inappropriate deposition of fibrillar collagens, impaired cardiac function and, ultimately, heart failure.^{9, 10}Autophagy is necessary for cellular homeostasis and is involved in organelle and protein turnover.^{11, 12, 13, 14} Autophagy aids in cell survival by providing primary materials, for example, amino acids and fatty acids for anabolic pathways during starvation conditions.^{15, 16} Alternatively, autophagy may be associated with apoptosis through autodigestive cellular processes, cellular infection with pathogens or extracellular stimuli.^{17, 18, 19, 20} The overall control of cardiac fibrosis is likely due to the complex functioning of an array of regulatory factors, but to date, there is little evidence linking autophagy with fibrogenesis in cardiac tissue.^{11, 12, 13, 14, 15, 16, 17, 18, 21, 22}Recent studies have demonstrated that TGF-β₁ may not only promote autophagy in mouse fibroblasts and human tubular epithelial kidney cells^{15, 23, 24} but can also inhibit this process in fibroblasts extracted from human patients with idiopathic pulmonary fibrosis.²⁵ Moreover, it has recently been reported that autophagy can negatively¹⁵ and positively^{25, 26, 27} regulate the fibrotic process in different model cell systems. In this study, we have explored the putative link between autophagy and TGF-β₁-induced fibrogenesis in human atrial myofibroblasts (hATMyofbs) and in a model of MI rat heart.     

Double-edged-sword effect of IL-1β on the osteogenesis of periodontal ligament stem cells via crosstalk between the NF-κB,MAPK and BMP/Smad signaling pathways

Microenvironmental conditions can interfere with the functional role and differentiation of mesenchymal stem cells (MSCs). Recent studies suggest that an inflammatory microenvironment can significantly impact the osteogenic potential of periodontal ligament stem cells (PDLSCs), but the precise effects and mechanisms involved remain unclear. Here, we show for the first time that interleukin-1β (IL-1β) has dual roles in the osteogenesis of PDLSCs at concentrations ranging from physiologically healthy levels to those found in chronic periodontitis. Low doses of IL-1β activate the BMP/Smad signaling pathway to promote the osteogenesis of PDLSCs, but higher doses of IL-1β inhibit BMP/Smad signaling through the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling, inhibiting osteogenesis. These results demonstrate that crosstalk between NF-κB, MAPK and BMP/Smad signaling mediates this dual effect of IL-1β on PDLSCs. We also show that the impaired osteogenesis of PDLSCs results in more inflammatory cytokines and chemokines being released, inducing the chemotaxis of macrophages, which further clarifies the role of PDLSCs in the pathogenesis of periodontitis.Approximately 90% of the population suffers from periodontitis,^{1, 2} which is characterized by chronic bacterial infections in the supporting structures of the teeth and a homeostatic imbalance between two coupled process in the periodontal system – bone resorption by osteoclasts and bone formation by osteoblasts. This disease involves interactions with bacterial products, numerous cell populations and different inflammatory mediators, and it can lead to tooth loss in adults.^{1, 2}Periodontal ligament stem cells (PDLSCs), a newly recognized sub-population of mesenchymal stem cells (MSCs), have attracted increasing attention in relation to their multipotency. As PDLSCs can easily be obtained from periodontal tissue, they are considered important for prospective cell-based therapies. Recently, PDLSCs have been shown to migrate to the site of periodontal lesions and to mediate periodontal regeneration.^{3, 4, 5} However, recent studies have found that the osteogenic capacity of stem cells is impaired in inflammatory microenvironments^6,7 and that there are complex interactions between stem cells and the microenvironment under pathological conditions. Our previous studies found that disrupted and disease-associated microenvironments could influence the characteristics and functions of MSCs.8-10 Additionally, some studies have indicated that MSCs act in an immunomodulatory manner to regulate the function and chemotaxis of immune cells and that environmental factors may determine which immunomodulatory pathways are operational in MSCs.¹¹ Thus, we assume that the mutual interactions between stem cells and inflammatory microenvironments are crucial to harnessing the regenerative potential of PDLSCs for therapeutic use.Interleukin-1 (IL-1) is a pleiotropic cytokine and a central mediator of innate immunity and inflammation.¹² In clinical studies, IL-1β has been found in increased concentrations in gingival crevicular fluid (GCF) and at sites of periodontal damage,^{13, 14} and levels of IL-1β have been reported to decrease after periodontal treatment.^{15, 16} Compared with levels at healthy sites, local IL-1β and tumor necrosis factor-α (TNF-α) levels in the microenvironments of chronic periodontitis have been found to be significantly elevated and to be associated with periodontal tissue destruction.17–19 IL-1 stimulates bone resorption by promoting osteoclast activation17^,20^,21 and mediates the osteoclastogenic effects of TNF-α by enhancing the expression of RANKL.15 In inflammatory microenvironments, IL-1 and TNF have a prominent role in the pathogenesis of periodontitis.19 Although TNF-α has activity similar to that of IL-1β, IL-1β is present at higher levels in inflamed gingival tissues, and its expression is limited to the connective tissue layer.22 Multiple studies have investigated the effect of IL-1β on osteoblast differentiation,^{23, 24} but conflicting data has been presented and the underlying mechanism of its effects remains unclear.25 A previous study has shown that the concentration of IL-1β in GCF is 145±167 pg/ml in healthy subjects and 6452±2289 pg/ml in patients with chronic periodontitis.26 In this study, we mimicked an inflammatory microenvironment using IL-1β at different concentrations that ranged from healthy physiological levels to those observed in the GCF in cases of chronic periodontitis26 and tried to establish an in vitro osteogenesis model to investigate the effects of different doses of IL-1β on PDLSCs.Previously, it has been reported that the nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways have crucial roles in the regulation of inflammation and bone metabolism.²⁷²⁸ In addition, the BMP/Smad signaling pathways have important roles in the regulation of osteoblast differentiation.29 However, the roles these signaling pathways have in the osteogenesis of MSCs in inflammatory microenvironments remain unclear. In the present study, we investigated the interactions of BMP/Smad, MAPK and NF-κB signaling pathways in mediating the IL-1β-regulated osteogenic differentiation of PDLSCs. Because the resident periodontal cells can produce various inflammatory mediators that induce inflammatory cells to invade the tissue and affect bone resorption,30 we further examined the role of PDLSCs in the pathogenesis of periodontitis by determining the production of inflammatory cytokines and chemokines by PDLSCs in which osteogenesis was inhibited by IL-1β.     

Pharmacological targeting of guanosine monophosphate synthase suppresses melanoma cell invasion and tumorigenicity

Malignant melanoma possesses one of the highest metastatic potentials among human cancers. Acquisition of invasive phenotypes is a prerequisite for melanoma metastases. Elucidation of the molecular mechanisms underlying melanoma invasion will greatly enhance the design of novel agents for melanoma therapeutic intervention. Here, we report that guanosine monophosphate synthase (GMPS), an enzyme required for the de novo biosynthesis of GMP, has a major role in invasion and tumorigenicity of cells derived from either BRAF^V600E or NRAS^Q61R human metastatic melanomas. Moreover, GMPS levels are increased in metastatic human melanoma specimens compared with primary melanomas arguing that GMPS is an attractive candidate for anti-melanoma therapy. Accordingly, for the first time we demonstrate that angustmycin A, a nucleoside-analog inhibitor of GMPS produced by Streptomyces hygroscopius efficiently suppresses melanoma cell invasion in vitro and tumorigenicity in immunocompromised mice. Our data identify GMPS as a powerful driver of melanoma cell invasion and warrant further investigation of angustmycin A as a novel anti-melanoma agent.Malignant melanoma is one of the most aggressive types of human cancers. Its ability to metastasize in combination with resistance to conventional anticancer chemotherapy makes melanoma extremely difficult to cure, and the median survival of patients with metastatic melanoma is 8.5 months.^{1, 2, 3} A better understanding of the biology behind melanoma aggressiveness is imperative to facilitate the development of novel anti-melanoma strategies.Melanoma and other cancers cells have been shown to strongly rely on de novo nucleotide biosynthesis^{4, 5} and often overexpress several biosynthetic enzymes involved in these pathways.^{6, 7, 8, 9} Recently, we have identified a fundamental connection between melanoma invasion and biosynthesis of guanylates,⁸ suggesting that distortion of the guanylate metabolism facilitates melanoma progression.Guanosine monophosphate reductase (GMPR) reduces GMP to one of its precursors, inosine monophosphate (IMP), and depletes intracellular GTP pools (Figure 1a). We have recently demonstrated that GMPR suppresses melanoma cell invasion and growth of human melanoma cell xenografts. These findings tightly linked guanylate production to the invasive potential of melanoma cells.⁸Open in a separate windowFigure 1GMPS contributes to the invasive capability of melanoma cells. (a) Simplified schematic of the metabolic pathway for guanylates production. (b) SK-Mel-103 and SK-Mel-28 cells were transduced with a control vector or two independent shRNAs to GMPS and tested for invasion through Matrigel (left panel). Where indicated, cells were incubated with 100 μM guanosine for 24 h before the assay and guanosine supplementation was maintained throughout the experimental procedure. The data represent the average ± S.E.M. of at least two independent experiments. GMPS suppression was verified by immunoblotting (right panel). (c) Cells transduced as in (a) were plated on coverslips coated with FITC-conjugated gelatin. After 16 h cells were fixed with 4% PFA and stained for actin (rhodamine-conjugated phallodin) and nuclei (Hoechst). Where indicated, cells were incubated with 100 μM guanosine for 24 h before the assay and guanosine supplementation was maintained throughout the experimental procedure. At least 25 cells/sample were imaged to assess the number of cells with gelatin degradation. The data represent the average ± S.E.M. of two independent experiments. *P<0.05, **P<0.001 compared with control; ^#P<0.05, ^##P<0.001 compared with untreated cells. Statistics performed with Student''s t-Test. See also Supplementary Figure S1Of the several enzymes involved in guanylate biosynthesis, inositol monophosphate dehydrogenases 1 and 2 (IMPDH-1, -2), functional antagonists of GMPR (Figure 1a), have been targeted clinically with several drugs including the most specific one, mycophenolic acid (MPA) and its salt, mycophenolate mofetil (MMF).^{10, 11, 12, 13} Nonetheless, prior studies demonstrated that MPA possesses poor anti-tumor activity,^{14, 15} and it is primarily used as an immunosuppressing agent in organ transplantation.^{10, 11, 12}GMP synthase (GMPS) is the other functional antagonist of GMPR. GMPS catalyzes the amination of xanitol monophosphate (XMP) to GMP to promote GTP synthesis (Figure 1a).^{16, 17} Most of the studies on GMPS have been performed in bacteria, yeast, and insects, where GMPS has been shown to have a key role in sporulation, pathogenicity, and axon guidance, respectively.^{18, 19, 20} Mammalian GMPS has been the subject of several studies addressing its unconventional (GMP-unrelated) roles in the regulation of activity of ubiquitin-specific protease 7 (USP7).^{21, 22, 23, 24} However, because of the newly revealed importance of guanylate metabolism in control of melanoma cell invasion and tumorigenicity,⁸ GMPS emerges as an attractive target for anti-cancer therapy.In the late 1950s, a specific inhibitor of bacterial GMPS, angustmycin A (also known as decoyinine), has been isolated from Streptomyces hygroscopius as a potential antibiotic with sporulation-inducing activity in Bacillus subtilis.^{25, 26, 27, 28, 29} Its anti-tumor activity has never been experimentally explored. In the current study, we investigated the role of GMPS in regulation of melanoma invasion and tumorigenicity, and explored the possibility of targeting GMPS by angustmycin A as a novel anti-melanoma strategy.     

Exploring the role of MKK7 in excitotoxicity and cerebral ischemia: a novel pharmacological strategy against brain injury

Excitotoxicity following cerebral ischemia elicits a molecular cascade, which leads to neuronal death. c-Jun-N-terminal kinase (JNK) has a key role in excitotoxic cell death. We have previously shown that JNK inhibition by a specific cell-permeable peptide significantly reduces infarct size and neuronal death in an in vivo model of cerebral ischemia. However, systemic inhibition of JNK may have detrimental side effects, owing to blockade of its physiological function. Here we designed a new inhibitor peptide (growth arrest and DNA damage-inducible 45β (GADD45β-I)) targeting mitogen-activated protein kinase kinase 7 (MKK7), an upstream activator of JNK, which exclusively mediates JNK''s pathological activation. GADD45β-I was engineered by optimizing the domain of the GADD45β, able to bind to MKK7, and by linking it to the TAT peptide sequence, to allow penetration of biological membranes. Our data clearly indicate that GADD45β-I significantly reduces neuronal death in excitotoxicity induced by either N-methyl-D-aspartate exposure or by oxygen–glucose deprivation in vitro. Moreover, GADD45β-I exerted neuroprotection in vivo in two models of ischemia, obtained by electrocoagulation and by thromboembolic occlusion of the middle cerebral artery (MCAo). Indeed, GADD45β-I reduced the infarct size when injected 30 min before the lesion in both models. The peptide was also effective when administrated 6 h after lesion, as demonstrated in the electrocoagulation model. The neuroprotective effect of GADD45β-I is long lasting; in fact, 1 week after MCAo the infarct volume was still reduced by 49%. Targeting MKK7 could represent a new therapeutic strategy for the treatment of ischemia and other pathologies involving MKK7/JNK activation. Moreover, this new inhibitor can be useful to further dissect the physiological and pathological role of the JNK pathway in the brain.In many disorders of the nervous system, overactivation of N-methyl-D-aspartate (NMDA) receptors leads to neuronal death and consequent neurological impairment. NMDA-induced neuronal death, that is, excitotoxicity, has been implicated in many neurodegenerative diseases such as stroke, epilepsy, Alzheimer disease, spinal cord injury, traumatic brain injury, hearing loss, Parkinson''s and Huntington diseases.¹ However, the molecular mechanisms underlying excitotoxic neuronal death remain only partially understood.Excitotoxicity triggers complex signal transduction events that induce the neuronal death program. Among them, activation of the c-Jun N-terminal kinase (JNK) pathway has a key role.^{2, 3, 4, 5} There are only two direct upstream activators of JNK: mitogen-activated protein kinase kinase 4 and 7 (MKK4 and MKK7).^{6, 7} In some cell types, MKK4 activates JNK primarily in response to stress stimuli, whereas MKK7 signaling is triggered by release of inflammatory cytokines.^{8, 9, 10} In neurons, however, we showed that MKK7 is mainly responsible for JNK overactivation during excitotoxicity both in vitro³ and in vivo following middle cerebral artery occlusion (MCAo).⁴ Conversely, MKK4 controls JNK physiological role and its activation is not affected by excitotoxic stimuli.³Inhibition of the JNK pathway by the specific JNK inhibitor peptide, D-JNKI1, has been proposed for the treatment of ischemia.² D-JNKI1 induces powerful neuroprotection in in vitro models of excitoxicity^{2, 11} and leads to a 93% reduction in the infarct size in rodent models of ischemia.^{2, 4, 12} Despite the potent and long-lasting neuroprotective effect of D-JNKI1, total inhibition of JNK is not deprived of negative side effects, as it regulates a variety of physiological events¹³ such as cell proliferation, survival and differentiation.¹³ For these reasons, MKK7 may represent a more attractive target for clinical application, as it activates JNK specifically after toxic stimuli. Thus, by targeting MKK7 the physiological role of JNK, regulated by MKK4, will be preserved.Here we designed a set of new cell-permeable inhibitor peptides able to block MKK7 activity and protect against excitotoxic death.We took advantage of the growth arrest and DNA damage-inducible 45β (GADD45β) ability to bind MKK7.^{9, 14, 15} GADD45β is involved in the control of cell stress responses in cell cycle, DNA repair and oncogenesis.^{9, 16} GADD45β binds tightly to MKK7 and inhibits its enzymatic activity¹⁵ by interacting with its catalytic domain.⁹ More importantly, GADD45β inhibition is MKK7-specific and has no effect on MKK4, MKK3/6 and MEK1/2 activity.⁹ The minimal essential domain of interaction between MKK7 and GADD45β has already been defined (GADD45β_60–86 and _69–86 sequences).¹⁵ We here used in silico approaches to design an effector peptide, based on the domain of GADD45β, and optimize its affinity for MKK7. We then linked the effector peptide to a TAT-cargo in order to penetrate neuronal plasma membrane.¹⁷ The selected cell-permeable MKK7 inhibitor peptide (GADD45β-l) confers neuroprotection in vitro against NMDA and oxygen–glucose deprivation (OGD) toxicity, as well as in vivo in two models of MCAo with a clinically relevant post-ischemic temporal window (6 h) at both 24 h and 1 week after lesion. These data shed light on a new approach for the treatment of ischemia.