Phosphorylation of CHIP at Ser20 by Cdk5 promotes tAIF-mediated neuronal death

Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase and its dysregulation is implicated in neurodegenerative diseases. Likewise, C-terminus of Hsc70-interacting protein (CHIP) is linked to neurological disorders, serving as an E3 ubiquitin ligase for targeting damaged or toxic proteins for proteasomal degradation. Here, we demonstrate that CHIP is a novel substrate for Cdk5. Cdk5 phosphorylates CHIP at Ser20 via direct binding to a highly charged domain of CHIP. Co-immunoprecipitation and ubiquitination assays reveal that Cdk5-mediated phosphorylation disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF) without affecting CHIP''s E3 ligase activity, resulting in the inhibition of CHIP-mediated degradation of tAIF. Lentiviral transduction assay shows that knockdown of Cdk5 or overexpression of CHIP^S20A, but not CHIP^WT, attenuates tAIF-mediated neuronal cell death induced by hydrogen peroxide. Thus, we conclude that Cdk5-mediated phosphorylation of CHIP negatively regulates its neuroprotective function, thereby contributing to neuronal cell death progression following neurotoxic stimuli.Cyclin-dependent kinase 5 (Cdk5) is a member of cyclin-dependent kinase that is crucial for the regulation of various post-mitotic cellular processes in the nervous system.^{1, 2, 3, 4} Although Cdk5 is activated upon direct interaction with a neural-specific activator p35 in normal condition,¹ it can be hyperactivated in numerous pathological conditions by the conversion of p35 to p25 via calpain.⁵ Therefore, dysregulation of Cdk5 activity has been linked to an array of neurodegenerative diseases.^{2, 5, 6} Consequently, Cdk5-mediated phosphorylation of several key intracellular substrates has received significant attention and has been identified to associate with the pathophysiology of neurodegeneration.^{6, 7}Recently, our laboratory made an attempt to identify the novel substrate of Cdk5 and we came across that Cdk5 interacts with C-terminus of Hsc70-interacting protein (CHIP). CHIP is composed of three major domains: (1) an amino-terminal three tetratricopeptide repeat (TPR) domain interacting with Hsc/Hsp70 and Hsp90, (2) a highly charged central domain with unknown functions and (3) a carboxyl-terminal U-box domain conferring E3 ubiquitin ligase activity.^{8, 9, 10} Based on these domains, it has been indicated that CHIP has a crucial role as a molecular co-chaperone in the maintenance of protein homeostasis during cellular stress and recovery.^{8, 11, 12, 13} Indeed, CHIP has been demonstrated to serve as a crucial catalyst for ubiquitination of Hsp70 client proteins targeting for proteasome-dependent degradation.¹¹ The function of CHIP with its molecular chaperones has been well documented to reduce cellular toxicity associated with several neurodegenerative diseases.^{11, 12, 14, 15, 16, 17, 18} However, the mechanisms underlying how function of CHIP is regulated and, if any, the pathophysiological cascades contributing to CHIP-mediated neurodegeneration are not fully elucidated yet.Here, we propose the molecular cascade in which Cdk5 phosphorylation of CHIP at Ser20 disrupts the interaction between CHIP and truncated apoptosis-inducing factor (tAIF), thereby contributes to escaping of tAIF from CHIP-mediated proteasome-dependent degradation, and eventually leads to tAIF-mediated neuronal cell death. We argue that this cascade explains one plausible mechanism as to how dysregulation of Cdk5 leads to neuronal death by negatively regulating CHIP-mediated degradation of toxic molecules.     

The Fcp1-Wee1-Cdk1 axis affects spindle assembly checkpoint robustness and sensitivity to antimicrotubule cancer drugs

To grant faithful chromosome segregation, the spindle assembly checkpoint (SAC) delays mitosis exit until mitotic spindle assembly. An exceedingly prolonged mitosis, however, promotes cell death and by this means antimicrotubule cancer drugs (AMCDs), that impair spindle assembly, are believed to kill cancer cells. Despite malformed spindles, cancer cells can, however, slip through SAC, exit mitosis prematurely and resist killing. We show here that the Fcp1 phosphatase and Wee1, the cyclin B-dependent kinase (cdk) 1 inhibitory kinase, play a role for this slippage/resistance mechanism. During AMCD-induced prolonged mitosis, Fcp1-dependent Wee1 reactivation lowered cdk1 activity, weakening SAC-dependent mitotic arrest and leading to mitosis exit and survival. Conversely, genetic or chemical Wee1 inhibition strengthened the SAC, further extended mitosis, reduced antiapoptotic protein Mcl-1 to a minimum and potentiated killing in several, AMCD-treated cancer cell lines and primary human adult lymphoblastic leukemia cells. Thus, the Fcp1-Wee1-Cdk1 (FWC) axis affects SAC robustness and AMCDs sensitivity.The spindle assembly checkpoint (SAC) delays mitosis exit to coordinate anaphase onset with spindle assembly. To this end, SAC inhibits the ubiquitin ligase Anaphase-Promoting Complex/Cyclosome (APC/C) to prevent degradation of the anaphase inhibitor securin and cyclin B, the major mitotic cyclin B-dependent kinase 1 (cdk1) activator, until spindle assembly.¹ However, by yet poorly understood mechanisms, exceedingly prolonging mitosis translates into cell death induction.^{2, 3, 4, 5, 6, 7} Although mechanistic details are still missing on how activation of cell death pathways is linked to mitosis duration, prolongation of mitosis appears crucial for the ability of antimicrotubule cancer drugs (AMCDs) to kill cancer cells.^{2, 3, 4, 5, 6, 7} These drugs, targeting microtubules, impede mitotic spindle assembly and delay mitosis exit by chronically activating the SAC. Use of these drugs is limited, however, by toxicity and resistance. A major mechanism for resistance is believed to reside in the ability of cancer cells to slip through the SAC and exit mitosis prematurely despite malformed spindles, thus resisting killing by limiting mitosis duration.^{2, 3, 4, 5, 6, 7} Under the AMCD treatment, cells either die in mitosis or exit mitosis, slipping through the SAC, without or abnormally dividing.^{2, 3, 4} Cells that exit mitosis either die at later stages or survive and stop dividing or proliferate, giving rise to resistance.^{2, 3, 4} Apart from a role for p53, what dictates cell fate is still unknown; however, it appears that the longer mitosis is protracted, the higher the chances for cell death pathway activation are.^{2, 3, 4, 5, 6, 7}Although SAC is not required per se for killing,⁶ preventing SAC adaptation should improve the efficacy of AMCD by increasing mitosis duration.^{2, 3, 4, 5, 6, 7} Therefore, further understanding of the mechanisms by which cells override SAC may help to improve the current AMCD therapy. Several kinases are known to activate and sustain SAC, and cdk1 itself appears to be of primary relevance.^{1, 8, 9} By studying mitosis exit and SAC resolution, we recently reported a role for the Fcp1 phosphatase to bring about cdk1 inactivation.^{10, 11} Among Fcp1 targets, we identified cyclin degradation pathway components, such as Cdc20, an APC/C co-activator, USP44, a deubiquitinating enzyme, and Wee1.^{10, 11} Wee1 is a crucial kinase that controls the G2 phase by performing inhibitory phosphorylation of cdk1 at tyr-15 (Y15-cdk1). Wee1 is also in a feedback relationship with cdk1 itself that, in turn, can phosphorylate and inhibit Wee1 in an autoamplification loop to promote the G2-to-M phase transition.¹² At mitosis exit, Fcp1 dephosphorylated Wee1 at threonine 239, a cdk1-dependent inhibitory phosphorylation, to dampen down the cdk1 autoamplification loop, and Cdc20 and USP44, to promote APC/C-dependent cyclin B degradation.^{10, 11, 12} In this study we analysed the Fcp1 relevance in SAC adaptation and AMCD sensitivity.     

Cell cycle-dependent Cdc25C phosphatase determines cell survival by regulating apoptosis signal-regulating kinase 1

Cdc25C (cell division cycle 25C) phosphatase triggers entry into mitosis in the cell cycle by dephosphorylating cyclin B-Cdk1. Cdc25C exhibits basal phosphatase activity during interphase and then becomes activated at the G₂/M transition after hyperphosphorylation on multiple sites and dissociation from 14-3-3. Although the role of Cdc25C in mitosis has been extensively studied, its function in interphase remains elusive. Here, we show that during interphase Cdc25C suppresses apoptosis signal-regulating kinase 1 (ASK1), a member of mitogen-activated protein (MAP) kinase kinase kinase family that mediates apoptosis. Cdc25C phosphatase dephosphorylates phospho-Thr-838 in the activation loop of ASK1 in vitro and in interphase cells. In addition, knockdown of Cdc25C increases the activity of ASK1 and ASK1 downstream targets in interphase cells, and overexpression of Cdc25C inhibits ASK1-mediated apoptosis, suggesting that Cdc25C binds to and negatively regulates ASK1. Furthermore, we showed that ASK1 kinase activity correlated with Cdc25C activation during mitotic arrest and enhanced ASK1 activity in the presence of activated Cdc25C resulted from the weak association between ASK1 and Cdc25C. In cells synchronized in mitosis following nocodazole treatment, phosphorylation of Thr-838 in the activation loop of ASK1 increased. Compared with hypophosphorylated Cdc25C, which exhibited basal phosphatase activity in interphase, hyperphosphorylated Cdc25C exhibited enhanced phosphatase activity during mitotic arrest, but had significantly reduced affinity to ASK1, suggesting that enhanced ASK1 activity in mitosis was due to reduced binding of hyperphosphorylated Cdc25C to ASK1. These findings suggest that Cdc25C negatively regulates proapoptotic ASK1 in a cell cycle-dependent manner and may play a role in G₂/M checkpoint-mediated apoptosis.Cell division cycle 25 (Cdc25) phosphatases are dual-specificity phosphatases involved in cell cycle regulation. By removing inhibitory phosphate groups from phospho-Thr and phospho-Tyr residues of cyclin-dependent kinases (CDKs),¹ Cdc25 proteins regulate cell cycle progression in S phase and mitosis. In mammals, three isoforms of Cdc25 phosphatases have been reported: Cdc25A, which controls the G₁/S transition;^{2, 3} Cdc25B, which is a mitotic starter;⁴ and Cdc25C, which controls the G₂/M phase.⁵ Overexpression of Cdc25 phosphatases is frequently associated with various cancers.⁶ Upon exposure to DNA-damaging reagents like UV radiation or free oxygen radicals, Cdc25 phosphatases are key targets of the checkpoint machinery, resulting in cell cycle arrest and apoptosis. The 14-3-3 proteins bind to phosphorylated Ser-216 of Cdc25C and induce Cdc25C export from the nucleus during interphase in response to DNA damage,^{7, 8} but they have no apparent effect on Cdc25C phosphatase activity.^{9, 10} In addition, hyperphosphorylation of Cdc25C correlates to its enhanced phosphatase activity.¹¹ Most studies with Cdc25C have focused on its role in mitotic progression. However, the role of Cdc25C is not clear when it is sequestered in the cytoplasm by binding to 14-3-3.Apoptosis signal-regulating kinase 1 (ASK1), also known as mitogen-activated protein kinase kinase kinase 5 (MAPKKK5), is a ubiquitously expressed enzyme with a molecular weight of 170 kDa. The kinase activity of ASK1 is stimulated by various cellular stresses, such as H₂O₂,^{12, 13} tumor necrosis factor-α (TNF-α),¹⁴ Fas ligand,¹⁵ serum withdrawal,¹³ and ER stress.¹⁶ Stimulated ASK1 phosphorylates and activates downstream MAP kinase kinases (MKKs) involved in c-Jun N-terminal kinase (JNK) and p38 pathways.^{17, 18, 19} Phosphorylation and activation of ASK1 can induce apoptosis, differentiation, or other cellular responses, depending on the cell type. ASK1 is regulated either positively or negatively depending on its binding proteins.^{12, 13, 15, 18, 19, 20, 21, 22, 23, 24, 25}ASK1 is regulated by phosphorylation at several Ser/Thr/Tyr residues. Phosphorylation at Thr-838 leads to activation of ASK1, whereas phosphorylation at Ser-83, Ser-967, or Ser-1034 inactivates ASK1.^{24, 26, 27, 28} ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site to inhibit ASK1.²⁴ Phosphorylation at Ser-83 is known to be catalyzed by Akt or PIM1.^{27, 29} Oligomerization-dependent autophosphorylation at Thr-838, which is located in the activation loop of the kinase domain, is essential for ASK1 activation.^{14, 18, 30} Phosphorylation at Tyr-718 by JAK2 induces ASK1 degradation.³¹ Several phosphatases that dephosphorylate some of these sites have been identified. Serine/threonine protein phosphatase type 5 (PP5) and PP2C dephosphorylate phosphorylated (p)-Thr-838,^{28, 32} whereas PP2A and SHP2 dephosphorylate p-Ser-967 and p-Tyr-718, respectively.^{31, 33} Little is known about the kinase or phosphatase that regulates phosphorylation at Ser-1034. Although ASK1 phosphorylation is known to be involved in the regulation of apoptosis, only a few reports show that ASK1 phosphorylation or activity is dependent on the cell cycle.^{21, 34}In this study, we examined the functional relationship between Cdc25C and ASK1 and identified a novel function of Cdc25C phosphatase that can dephosphorylate and inhibit ASK1 in interphase but not in mitosis. Furthermore, we demonstrated that Cdc25C phosphorylation status plays a critical role in the interaction with and the activity of ASK1. These results reveal a novel regulatory function of Cdc25C in the ASK1-mediated apoptosis signaling pathway.     

The plant metacaspase AtMC1 in pathogen-triggered programmed cell death and aging: functional linkage with autophagy

Autophagy is a major nutrient recycling mechanism in plants. However, its functional connection with programmed cell death (PCD) is a topic of active debate and remains not well understood. Our previous studies established the plant metacaspase AtMC1 as a positive regulator of pathogen-triggered PCD. Here, we explored the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered PCD and aging. We observed that autophagy acts as a positive regulator of pathogen-triggered PCD in a parallel pathway to AtMC1. In addition, we unveiled an additional, pro-survival homeostatic function of AtMC1 in aging plants that acts in parallel to a similar pro-survival function of autophagy. This novel pro-survival role of AtMC1 may be functionally related to its prodomain-mediated aggregate localization and potential clearance, in agreement with recent findings using the single budding yeast metacaspase YCA1. We propose a unifying model whereby autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants, when these functions are not masked by the cumulative stresses of aging, and negatively regulating senescence in older plants.An emerging theme in cell death research is that cellular processes thought to be regulated by linear signaling pathways are, in fact, complex. Autophagy, initially considered merely a nutrient recycling mechanism necessary for cellular homeostasis, was recently shown to regulate cell death, mechanistically interacting with components that control apoptosis. Deficient autophagy can result in apoptosis^{1, 2, 3} and autophagy hyper-activation can also lead to programmed cell death (PCD).⁴ In addition, the pro-survival function of autophagy is mediated by apoptosis inhibition and apoptosis mediates autophagy, although this cross-regulation is not fully understood.⁵In plants, autophagy can also have both pro-survival and pro-death functions. Autophagy-deficient plants exhibit accelerated senescence,^{6, 7, 8} starvation-induced chlorosis,^{6, 7, 9} hypersensitivity to oxidative stress¹⁰ and endoplasmic reticulum stress.¹¹ Further, autophagy-deficient plants cannot limit the spread of cell death after infection with tissue-destructive microbial infections.^{12, 13} The plant phytohormone salicylic acid (SA) mediates most of these phenotypes.⁸ Autophagy has an essential, pro-survival role in situations where there is an increasing load of damaged proteins and organelles that need to be eliminated, that is, during aging or stress. Autophagy has an opposing, pro-death role during developmentally regulated cell death^{14, 15} or during the pathogen-triggered hypersensitive response PCD (hereafter, HR) that occurs locally at the site of attempted pathogen attack.^{16, 17} The dual pro-death/pro-survival functions of plant autophagy remain a topic of active debate.Also under scrutiny are possible novel functions of caspases and caspase-like proteins as central regulators of pro-survival processes. Caspases were originally defined as executioners of PCD in animals, but increasing evidence indicates that several caspases have non-apoptotic regulatory roles in cellular differentiation, motility and in the mammalian immune system.^{18, 19, 20}Yeast, protozoa and plants do not have canonical caspases, despite the occurrence of morphologically heterogeneous PCDs.²¹ More than a decade ago, distant caspase homologs termed metacaspases were identified in these organisms using structural homology searches.²² Metacaspases were classified into type I or type II metacaspases based on the presence or absence of an N-terminal prodomain, reminiscent of the classification in animals into initiator/inflammatory or executioner caspases, respectively. Despite the architectural analogy between caspases and metacaspases, differences in their structure, function, activation and mode of action exist.^{23, 24, 25}Metacaspases mediate PCD in yeast,^{26, 27, 28, 29, 30, 31} leishmania,^{32, 33} trypanosoma³⁴ and plants.²⁴ We demonstrated that two type I metacaspases, AtMC1 and AtMC2, antagonistically regulate HR in Arabidopsis thaliana.³⁵ Our work showed that AtMC1 is a positive regulator of HR and that this function is mediated by its catalytic activity and negatively regulated by the AtMC1 N-terminal prodomain. AtMC2 antagonizes AtMC1-mediated HR.Besides AtMC2, new examples of metacaspases with a pro-life/non-PCD role are emerging. Protozoan metacaspases are involved in cell cycle dynamics^{34, 36, 37, 38} and cell proliferation.³⁹ The yeast metacaspase Yca1 alters cell cycle dynamics⁴⁰ and interestingly, is required for clearance of insoluble protein aggregates, thus contributing to yeast fitness.⁴¹Here, we explore the linkage between plant autophagy and AtMC1 function in the context of pathogen-triggered HR and aging. Our data support a model wherein autophagy and AtMC1 are part of parallel pathways, both positively regulating HR cell death in young plants and negatively regulating senescence in older plants.     

Dysferlin regulates cell membrane repair by facilitating injury-triggered acid sphingomyelinase secretion

Dysferlin deficiency compromises the repair of injured muscle, but the underlying cellular mechanism remains elusive. To study this phenomenon, we have developed mouse and human myoblast models for dysferlinopathy. These dysferlinopathic myoblasts undergo normal differentiation but have a deficit in their ability to repair focal injury to their cell membrane. Imaging cells undergoing repair showed that dysferlin-deficit decreased the number of lysosomes present at the cell membrane, resulting in a delay and reduction in injury-triggered lysosomal exocytosis. We find repair of injured cells does not involve formation of intracellular membrane patch through lysosome–lysosome fusion; instead, individual lysosomes fuse with the injured cell membrane, releasing acid sphingomyelinase (ASM). ASM secretion was reduced in injured dysferlinopathic cells, and acute treatment with sphingomyelinase restored the repair ability of dysferlinopathic myoblasts and myofibers. Our results provide the mechanism for dysferlin-mediated repair of skeletal muscle sarcolemma and identify ASM as a potential therapy for dysferlinopathy.Dysferlinopathy is a progressive muscle wasting disease, which is classified as limb-girdle muscular dystrophy type 2B (LGMD2B) or Miyoshi muscular dystrophy 1, based on its muscle involvement.^{1, 2} Dysferlin deficit leads to altered vesicle formation and trafficking,^{3, 4} poor repair of injured cell membranes,^{5, 6} and increased muscle inflammation.^{7, 8} Dysferlin contains C2 domains that are found in Ca²⁺-dependent membrane fusion proteins such as synaptotagmins.⁹ Thus, dysferlin is thought to regulate muscle function by regulating vesicle trafficking and fusion.^{10, 11, 12, 13} Dysferlin deficiency has also been implicated in conflicting reports regarding the fusion ability of dysferlinopathic myoblasts.^{4, 14, 15, 16} With such diverse roles for dysferlin, the mechanism through which dysferlin deficiency results in muscle pathology is unresolved. As skeletal muscle-specific re-expression of dysferlin rescues all dysferlinopathic pathologies,^{17, 18} myofiber repair has been suggested to be the unifying deficit underlying muscle pathology in dysferlinopathy.¹⁹ Repair of injured cell membranes requires subcellular compartments, which in mammalian cells include lysosomes,¹¹ enlargeosomes,²⁰ caveolae,²¹ dysferlin-containing vesicles,⁵ and mitochondria.²²Cells from muscular dystrophy patients that have normal dysferlin expression exhibit normal lysosome and enlargeosome exocytosis.²³ However, dysferlinopathic muscle cells exhibit enlarged LAMP2-positive lysosomes, reduced fusion of early endosomes, altered expression of proteins regulating late endosome/lysosome fusion, and reduced injury-triggered cell-surface levels of LAMP1.^{4, 11, 12} In non-muscle cells, lack of dysferlin reduces lysosomal exocytosis.²⁴ These findings implicate lysosomes in dysferlin-mediated muscle cell membrane repair. In one model for lysosome-mediated cell membrane repair, Ca²⁺ triggers vesicle–vesicle fusion near the site of injury, forming ‘membrane patch'', which fuses to repair the wounded cell membrane.^{25, 26, 27, 28} In another model, lysosome exocytosis following cell membrane injury by pore-forming toxins leads to secretion of the lysosomal enzyme acid sphingomyelinase (ASM), which causes endocytosis of pores in the damaged cell membranes.^{21, 29, 30} Both these models have been suggested to be involved in the repair of injured muscle cells.^{21, 28}To examine the muscle cell pathology in dysferlinopathy, we have developed dysferlinopathic mouse and human models. Use of these models shows that a lack of dysferlin does not alter myogenic differentiation but causes poor repair of even undifferentiated muscle cells. We show that dysferlin is required for tethering lysosomes to the cell membrane. Fewer lysosomes at the cell membrane in dysferlinopathic cells results in slow and reduced lysosome exocytosis following injury. This reduction in exocytosis reduces injury-triggered ASM secretion, which is responsible for the poor repair of dysferlinopathic muscle cells. Extracellular sphingomyelinase (SM) fully rescues the repair deficit in dysferlinopathic cells and mouse myofibers, offering a potential drug-based therapy for dysferlinopathy.     

Mps1 kinase regulates tumor cell viability via its novel role in mitochondria

Targeting mitotic kinase monopolar spindle 1 (Mps1) for tumor therapy has been investigated for many years. Although it was suggested that Mps1 regulates cell viability through its role in spindle assembly checkpoint (SAC), the underlying mechanism remains less defined. In an endeavor to reveal the role of high levels of mitotic kinase Mps1 in the development of colon cancer, we unexpectedly found the amount of Mps1 required for cell survival far exceeds that of maintaining SAC in aneuploid cell lines. This suggests that other functions of Mps1 besides SAC are also employed to maintain cell viability. Mps1 regulates cell viability independent of its role in cytokinesis as the genetic depletion of Mps1 spanning from metaphase to cytokinesis affects neither cytokinesis nor cell viability. Furthermore, we developed a single-cycle inhibition strategy that allows disruption of Mps1 function only in mitosis. Using this strategy, we found the functions of Mps1 in mitosis are vital for cell viability as short-term treatment of mitotic colon cancer cell lines with Mps1 inhibitors is sufficient to cause cell death. Interestingly, Mps1 inhibitors synergize with microtubule depolymerizing drug in promoting polyploidization but not in tumor cell growth inhibition. Finally, we found that Mps1 can be recruited to mitochondria by binding to voltage-dependent anion channel 1 (VDAC1) via its C-terminal fragment. This interaction is essential for cell viability as Mps1 mutant defective for interaction fails to main cell viability, causing the release of cytochrome c. Meanwhile, deprivation of VDAC1 can make tumor cells refractory to loss of Mps1-induced cell death. Collectively, we conclude that inhibition of the novel mitochondrial function Mps1 is sufficient to kill tumor cells.Massive chromosome missegregation induces cell death as observed by Theodor Boveri in the early 1900s.¹ However, the underlying mechanism remains elusive. The spindle assembly checkpoint (SAC) is a dominant machine monitoring chromosomal segregation during mitosis by delaying the onset of anaphase until all chromosomes are properly captured by microtubules. The SAC consists of kinetochore association sensors, including Mps1 (monopolar spindle 1), Bub1 (budding uninhibited by benzimidazole 1 homolog) and Aurora B; a signaling transducer termed the mitotic checkpoint complex (MCC), including CDC20 (cell division cycle 20), BubR1 (Bub1-related kinase), Bub3 (budding uninhibited by benzimidazole 3 homolog) and Mad2 (mitotic arrest deficient-like 2); and an effector APC/C (anaphase-promoting complex/cyclosome) that is inhibited by MCC in response to an active SAC.² Loss of SAC by inactivation of checkpoint sensors or signaling transducers elicits massive chromosome missegregation, induces severe gain or loss of chromosomes and eventually causes cell death.^{3, 4, 5, 6} Meanwhile, a weakened SAC due to the haploinsufficiency of the checkpoint proteins Mad1, Mad2, Bub1, BubR1 and CENP-E (centromere protein E) does not cause cell death but facilitates tumorigenesis.^{7, 8, 9, 10, 11} These studies suggest that the fate of these cells is dependent on their respective degree of SAC deficiency. Notably, in these studies SAC proteins were constitutively disturbed, raising the possibility that other signaling pathways could be affected as SAC proteins have functions beyond SAC regulation.^{12, 13, 14}Mps1 is an essential component of SAC that senses SAC signal by promoting MCC formation via kinetochore recruitment of Mad2, CENP-E and Knl1 (kinetochore-null protein 1).^{15, 16, 17, 18, 19} Recent studies show that Mps1 can discriminate between on or off SAC signaling by binding to NDC80c via the motif that associates microtubules.^{20, 21} Following SAC, Mps1 is involved in regulating chromosome alignment by phosphorylating Borealin, a component of chromosomal passenger complex (CPC).^{22, 23} In addition, Mps1 plays multiple roles beyond mitosis, including centrosome duplication, cytokinesis, ciliogenesis and DNA damage response.^{18, 24, 25, 26, 27, 28} Mps1 is indispensable for cell survival as loss of Mps1 function by specific siRNA or Mps1 kinase inhibitors causes significant cell death; it has been proposed that Mps1 regulates this process through its roles in SAC.^{29, 30, 31}Mps1 kinase is overexpressed in a variety of tumor types.^{32, 33, 34, 35} In breast cancer, high levels of Mps1 correlate with tumor grades; reducing Mps1 level induces massive apoptosis but allows a selective survival of tumor cells with less aneuploidy.³² Our recent results in colon cancer cells showed that overexpression of Mps1 facilitate the survival of tumor cells with higher aneuploidy by decreasing SAC threshold.³⁵ To further uncover the roles of high levels of Mps1 in tumorigenesis, we examined Mps1 levels in various stages of colon cancer tissues and found that Mps1 level peaks in tissues at stage II, at which stage tumor cells encounter various survival stresses, including genome instability. Aneuploid colon cancer cell lines bear higher levels of Mps1 than diploid cell lines and the amount of Mps1 required for cell survival is far more than that of maintaining SAC, suggesting that other functions of Mps1 are also employed to maintain cell viability. Short-term inhibition of Mps1 activity in mitosis with inhibitors at a dose of more than SAC depletion is sufficient to cause dividing cell death and increase mitochondrial fragmentation simultaneously. Finally, we found that Mps1 can regulate the release of cytochrome c by associating with mitochondrial protein VDAC1 (voltage-dependent anion channel 1). Based on these findings, we postulated that high levels of Mps1 contribute to survival of aneuploid cancer cells via its roles in SAC and mitochondria.     

Antitumour potential of BPT: a dual inhibitor of cdk4 and tubulin polymerization

The marine natural product fascaplysin (1) is a potent Cdk4 (cyclin-dependent kinase 4)-specific inhibitor, but is toxic to all cell types possibly because of its DNA-intercalating properties. Through the design and synthesis of numerous fascaplysin analogues, we intended to identify inhibitors of cancer cell growth with good therapeutic window with respect to normal cells. Among various non-planar tryptoline analogues prepared, N-(biphenyl-2-yl) tryptoline (BPT, 6) was identified as a potent inhibitor of cancer cell growth and free from DNA-binding properties owing to its non-planar structure. This compound was tested in over 60 protein kinase assays. It displayed inhibition of Cdk4-cyclin D1 enzyme in vitro far more potently than many other kinases including Cdk family members. Although it blocks growth of cancer cells deficient in the mitotic-spindle checkpoint at the G₀/G₁ phase of the cell cycle, the block occurs primarily at the G₂/M phase. BPT inhibits tubulin polymerization in vitro and acts as an enhancer of tubulin depolymerization of paclitaxel-stabilized tubulin in live cells. Western blot analyses indicated that, in p53-positive cells, BPT upregulates the expression of p53, p21 and p27 proteins, whereas it downregulates the expression of cyclin B1 and Cdk1. BPT selectively kills SV40-transformed mouse embryonic hepatic cells and human fibroblasts rather than untransformed cells. BPT inhibited the growth of several human cancer cells with an IC₅₀ <1 μM. The pharmacokinetic study in BALB/c mice indicated good plasma exposure after intravenous administration. It was found to be efficacious at 1/10th the maximum-tolerated dose (1000 mg/kg) against human tumours derived from HCT-116 (colon) and NCI-H460 (lung) cells in SCID (severe-combined immunodeficient) mice models. BPT is a relatively better anticancer agent than fascaplysin with an unusual ability to block two overlapping yet crucial phases of the cell cycle, mitosis and G₀/G₁. Its ability to effectively halt tumour growth in human tumour-bearing mice would suggest that BPT has the potential to be a candidate for further clinical development.A link between development of human cancers and cellular pathways where the retinoblastoma protein (pRb) has a major role is well established.^{1, 2} One of the frequent events associated with human tumour progression is abnormality in the pathway that links pRb, p16^INK4A, cyclin D1 and Cdk4 (cyclin-dependent kinase 4).³ Cdk4 along with its activating cyclin partner D1 has a key role in cell cycle control.^{4, 5} The naturally occurring inhibitor of Cdk4-cyclin D1, p16^INK4a (p16), is a tumour supressor protein. Deletion or inactivating mutations in the p16 gene are observed in many human cancers.^{6, 7} The catalytic activity of Cdk4 depends on its activation by the protein cyclin D1, which is expressed during the G₀/G₁ phase of the cell cycle. Many cancers are characterised by abnormal overproduction of cyclin D1.^{8, 9} As Cdk4 inhibitors target a pathway that links pRb, p16INK4A, cyclin D1 and Cdk4, it makes inhibition of Cdk4-cyclin D1 enzyme a crucially important target for cancer chemotherapy.^{10, 11, 12, 13} However, Rb mutations, consistent with loss of Rb function, have been identified in a wide spectrum of tumours including osteosarcomas, small-cell lung carcinomas, breast carcinomas and others, and the Cdk4 inhibitors cannot inhibit such pathway involving Rb-mutated tumours.A number of potential anticancer agents that selectively modulate the activity of Cdk4-cyclin D1 in vitro have been reported.¹⁴ These molecules also show the genotypic consequences of Cdk4 enzyme inhibition at the cellular level, that is, growth inhibition of cancer cells in vitro, arrest of asynchronous cells at G₀/G₁ and prevention of pRb phosphorylation at Cdk4-specific serine residues.^{15, 16, 17} Usually, competing with ATP molecules for binding at the protein kinase active site is the normal mechanism by which most small molecules inhibit kinase enzyme activity. Successful attempts to identify selective Cdk4 inhibitors using structure-based chemical design and molecular modelling have been reported.^{18, 19, 20} Furthermore, the success of Cdk4 inhibitors at clinical stages^{21, 22, 23, 24, 25} has indicated it as a promising therapeutic target for anticancer drug discovery.¹⁴Fascaplysin (1), a natural product originally isolated from a marine sponge, specifically inhibits the Cdk4 enzyme.^{26, 27} It inhibits Cdk4-cyclin D1 with an IC₅₀ of ~0.35 μM and blocks growth of cancer cells at the G₀/G₁ phase of the cell cycle. Similar to cryptolepine and ellipticine,²⁸ fascaplysin is also a planar structure and thus it intercalates double-stranded (d-s) DNA and shows unusual toxicity at the cellular level. It has been suggested that fascaplysin''s planar structure is the possible explanation for its ability to intercalate d-s DNA and also its unusual toxicity at the cellular level. To overcome this unusual toxicity, recently we reported CA224 (2), a non-planar analogue of fascaplysin exhibiting selective Cdk4 inhibition with no DNA-intercalating property.²⁹ In continuation to these efforts, herein we report identification of tryptoline-based compounds CA198 (3), CA199 (4), CA211 (5) and N-(biphenyl-2-yl)-tryptoline (BPT, 6) as selective Cdk4 inhibitors with no DNA-intercalating property. Based on the molecular modelling design, a number of non-planar analogues of fascaplysin were synthesized. They show specificity towards Cdk4-cyclin D1 enzyme activity and blocks the growth of cancer cells at the G₀/G₁ phase. Although BPT was also designed using a homology model of Cdk4, based on the X-ray crystallographic structures of Cdk2, Cdk4 and Cdk6,^{30, 31, 32, 33} further investigations showed that BPT blocks growth of cells at the G₂/M phase, in a Cdk-independent manner, through inhibition of tubulin polymerization. BPT shows potent cytotoxicity in a panel of cancer cells and is efficacious against human tumours derived from HCT-116 and NCI-H460 cells in SCID mice models. Here, we present the biological activity of BPT in detail. BPT (6) was synthesized using a one-step procedure by coupling tryptoline with biphenyl 2-carboxylic acid. The chemical synthesis of BPT and chemical structures of 1-6 are shown in Figure 1.Open in a separate windowFigure 1(a) Chemical structures of fascaplysin (1), CA224 (2) and its tryptoline analogues 3–6. (b) Synthetic scheme for BPT (6). (c) Molecular modelling studies to understand Cdk4 selectivity versus Cdk2: interactions of cis/trans conformations of BPT with Cdk2 and Cdk4, respectively (orange conformation is with Cdk2 and green with Cdk4)     

Thrombospondin-1 signaling through CD47 inhibits cell cycle progression and induces senescence in endothelial cells

CD47 signaling in endothelial cells has been shown to suppress angiogenesis, but little is known about the link between CD47 and endothelial senescence. Herein, we demonstrate that the thrombospondin-1 (TSP1)-CD47 signaling pathway is a major mechanism for driving endothelial cell senescence. CD47 deficiency in endothelial cells significantly improved their angiogenic function and attenuated their replicative senescence. Lack of CD47 also suppresses activation of cell cycle inhibitors and upregulates the expression of cell cycle promoters, leading to increased cell cycle progression. Furthermore, TSP1 significantly accelerates replicative senescence and associated cell cycle arrest in a CD47-dependent manner. These findings demonstrate that TSP1-CD47 signaling is an important mechanism driving endothelial cell senescence. Thus, TSP1 and CD47 provide attractive molecular targets for treatment of aging-associated cardiovascular dysfunction and diseases involving endothelial dysregulation.Endothelial cell (EC) senescence is accompanied with vascular dysfunction, including arterial stiffening and remodeling,¹ impaired angiogenesis,^{2, 3} reduced endothelial repair capability and increased incidence of cardiovascular disease.^{4, 5, 6} Cellular senescence can occur in vivo or in vitro in response to various stressors,^{7, 8, 9, 10} leading to suppression of cell proliferation. EC senescence has been reported to contribute to the pathogenesis of age-associated vascular diseases, such as atherosclerosis.¹¹ Thus, further understanding the mechanisms of EC senescence may help to identify effective targets for antisenescence therapy and treatment aging-associated cardiovascular disorders.Previous studies have shown that the secreted matricellular protein thrombospondin-1 (TSP1) is as potent inhibitor of angiogenesis¹² and its antiangiogenic activity is mediated by its receptors, CD36^{13, 14} and CD47.^{15, 16} CD47 is a ubiquitously expressed transmembrane protein that serves as a ligand for signal regulatory protein-α and is a signaling receptor of TSP1. The TSP1-CD47 pathway has an important role in several fundamental cellular functions, including proliferation, apoptosis, inflammation and atherosclerotic response.¹⁷ Ligation of CD47 by TSP1 has been shown to inhibit nitric oxide (NO)/cGMP signaling in vascular cells, leading to suppression of angiogenic responses.¹⁶ Recently, it was reported that lack of CD47 expression in ECs may enable these cells to spontaneously gain characteristics of embryonic stem cells.¹⁸ However, the potential role of CD47 in regulation of EC senescence has not been well explored. The present study was initiated to determine the role and mechanisms of TSP1-CD47 signaling pathway in regulating cell cycle progression and replicative senescence of ECs.     

Adiponectin receptor-mediated signaling ameliorates cerebral cell damage and regulates the neurogenesis of neural stem cells at high glucose concentrations: an in vivo and in vitro study

In the central nervous system (CNS), hyperglycemia leads to neuronal damage and cognitive decline. Recent research has focused on revealing alterations in the brain in hyperglycemia and finding therapeutic solutions for alleviating the hyperglycemia-induced cognitive dysfunction. Adiponectin is a protein hormone with a major regulatory role in diabetes and obesity; however, its role in the CNS has not been studied yet. Although the presence of adiponectin receptors has been reported in the CNS, adiponectin receptor-mediated signaling in the CNS has not been investigated. In the present study, we investigated adiponectin receptor (AdipoR)-mediated signaling in vivo using a high-fat diet and in vitro using neural stem cells (NSCs). We showed that AdipoR1 protects cell damage and synaptic dysfunction in the mouse brain in hyperglycemia. At high glucose concentrations in vitro, AdipoR1 regulated the survival of NSCs through the p53/p21 pathway and the proliferation- and differentiation-related factors of NSCs via tailless (TLX). Hence, we suggest that further investigations are necessary to understand the cerebral AdipoR1-mediated signaling in hyperglycemic conditions, because the modulation of AdipoR1 might alleviate hyperglycemia-induced neuropathogenesis.Adiponectin secreted by the adipose tissue^{1, 2} exists in either a full-length or globular form.^{3, 4, 5, 6} Adiponectin can cross the blood–brain barrier, and various forms of adiponectin are found in the cerebrospinal fluid.^{7, 8, 9, 10, 11} Adiponectin exerts its effect by binding to the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2)^{12, 13} that have different affinities for the various circulating adiponectins.^{12, 14, 15, 16, 17} Several studies reported that both receptor subtypes are expressed in the central nervous system (CNS).^{7, 12, 18} As adiponectin modulates insulin sensitivity and inflammation,¹⁹ its deficiency induces insulin resistance and glucose intolerance in animals fed a high-fat diet (HFD).^{19, 20, 21} In addition, adiponectin can ameliorate the glucose homeostasis and increase insulin sensitivity.^{22, 23, 24} Adiponectin, which is the most well-known adipokine, acts mainly as an anti-inflammatory regulator,^{25, 26} and is associated with the onset of neurological disorders.²⁷ In addition, a recent study reported that adiponectin promotes the proliferation of hippocampal neural stem cells (NSCs).²⁸ Considering that adiponectin acts by binding to the adiponectin receptors, investigation of the adiponectin receptor-mediated signaling in the brain is crucial to understand the cerebral effects of adiponectin and the underlying cellular mechanisms.The prevalence of type II diabetes mellitus (DM2) and Alzheimer''s disease increases with aging.²⁹ According to a cross-sectional study, in people with DM2, the risk of dementia is 2.5 times higher than that in the normal population.^{30, 31} A study performed between 1980 and 2002 suggested that an elevated blood glucose level is associated with a greater risk for dementia in elderly patients with DM2.³² In addition, according to a 9-year-long longitudinal cohort study, the risk of developing Alzheimer''s disease was 65% higher in people with diabetes than in control subjects.³³ A community-based cohort study also reported that higher plasma glucose concentrations are associated with an increased risk for dementia, because the higher glucose level has detrimental effects on the brain.³¹ High blood glucose level causes mitochondria-dependent apoptosis,^{34, 35, 36} and aggravates diverse neurological functions.^{37, 38} Inflammation and oxidative stress, which are commonly observed in people with diabetes, inhibit neurogenesis.^{39, 40, 41} Similarly, neurogenesis is decreased in mice and rats with genetically induced type I diabetes.^{42, 43} In addition, diabetic rodents have a decreased proliferation rate of neural progenitors.^{43, 44} Furthermore, several studies suggested that an HFD leads to neuroinflammation, the impairment of synaptic plasticity, and cognitive decline.^{45, 46}Here, we investigated whether AdipoR1-mediated signaling is associated with cell death in the brain of mice on a HFD, and whether high glucose level modifies the proliferation and differentiation capacity of NSCs in vitro. Our study provides novel findings about the role of AdipoR1-mediated signaling in hyperglycemia-induced neuropathogenesis.     

Implication of different domains of the Leishmania major metacaspase in cell death and autophagy

Metacaspases (MCAs) are cysteine peptidases expressed in plants, fungi and protozoa, with a caspase-like histidine–cysteine catalytic dyad, but differing from caspases, for example, in their substrate specificity. The role of MCAs is subject to debate: roles in cell cycle control, in cell death or even in cell survival have been suggested. In this study, using a Leishmania major MCA-deficient strain, we showed that L. major MCA (LmjMCA) not only had a role similar to caspases in cell death but also in autophagy and this through different domains. Upon cell death induction by miltefosine or H₂O₂, LmjMCA is processed, releasing the catalytic domain, which activated substrates via its catalytic dyad His/Cys and a proline-rich C-terminal domain. The C-terminal domain interacted with proteins, notably proteins involved in stress regulation, such as the MAP kinase LmaMPK7 or programmed cell death like the calpain-like cysteine peptidase. We also showed a new role of LmjMCA in autophagy, acting on or upstream of ATG8, involving Lmjmca gene overexpression and interaction of the C-terminal domain of LmjMCA with itself and other proteins. These results allowed us to propose two models, showing the role of LmjMCA in the cell death and also in the autophagy pathway, implicating different protein domains.Apoptosis is, in most cases, associated with and depends on the activation of cys-dependent peptidases, named caspases.^{1, 2} Once activated, initiator caspases induce a proteolytic cascade via the activation of effector caspases that ultimately cleave numerous substrates, thereby causing the typical morphological features of apoptosis.^{3, 4} Despite their essential role in apoptosis, caspases are also involved in non-apoptotic events, including inflammation, cell proliferation, cell differentiation⁵ and the cell survival process autophagy, a major catabolic process in eukaryotic cells that allows cells to survive nutrient starvation due to engulfment of a portion of the cytoplasm by a specific membrane, delivery to lysosomes or vacuoles and digestion by hydrolytic enzymes.^{6, 7, 8, 9, 10} Plants, fungi and protozoa are devoid of caspases but express metacaspases (MCAs).¹¹MCAs are cysteine peptidases of the clan CD, family 14, with a caspase-like histidine–cysteine catalytic dyad.^{12, 13} However, besides their distant similarity to caspases,¹⁴ MCAs prefer arginine/lysine in the P1 position, whereas caspases prefer aspartic residues.^{15, 16} The role of MCAs in cell death is still enigmatic. For example, in the yeast Saccharomyces cerevisiae, YCA1 has a role in cell death,^{17, 18} whereas, although only partly dependent on its conserved catalytic cysteine, it also facilitates the removal of unfolded proteins, prolonging cellular life span.¹⁹ Similarly, some metacaspases have roles, outside of death, in stress acclimation pathways, as in Aspergillus fumigatus²⁰ or in the unicellular planctonic organisms diatoms.^{21, 22} In Arabidopsis thaliana, AtMC1 is a positive regulator of cell death and a survival factor for aging plants,²³ whereas AtMC2 negatively regulates cell death.²⁴ Trypanosoma brucei TbMCA2, TbMCA3 and TbMCA5 and Leishmania major MCA are involved in cell cycle regulation.^{25, 26}Leishmania are parasitic protozoa responsible for the neglected tropical disease leishmaniasis, transmitted to humans by the bite of the sand fly. In the insect, parasites proliferate as free-living flagellated forms called procyclic promastigotes within the midgut before differentiating into virulent metacyclic promastigotes and migrating to the proboscis.^{27, 28} In the mammalian host, promastigotes are taken up by macrophages and transform into amastigotes. Under a variety of stress stimuli, apoptosis-like morphological and biochemical features have been described in Leishmania, among which are cell shrinkage, chromatin condensation, DNA fragmentation or mitochondrial depolarization.^{29, 30, 31, 32, 33, 34, 35, 36, 37, 38} Despite the evidence of morphological and biochemical markers of cell death in dying Leishmania, very little is known about the cell death pathway and the implicated executioner proteins. Indeed, essential proteins involved in mammalian apoptosis, death receptors, small pro- and anti-apoptotic molecules and caspases, are apparently not encoded in the genome of Leishmania³⁹ and the role of Leishmania MCA in cell death is still controversial, certain authors suggesting a role as a negative regulator of intracellular amastigote proliferation, instead of having a caspase-like role in the execution of cell death.⁴⁰LmjMCA contains different domains: an N-terminal domain with a Mitochondrion Localization Signal (MLS),⁴¹ a caspase-like catalytic domain and a C-terminal proline-rich domain.⁴¹ On the basis of this domain structure, LmjMCA can be classified among the type I metacaspases,¹⁶ a subclass more generally defined in higher plants and characterized by the presence of an N-terminal prodomain and a short linker between the large and small subunits, as initiator caspases in metazoans.¹¹ Upon induction of cell death by heat shock, H₂O₂ or drugs like miltefosine or curcumin, LmjMCA is processed and the catalytic domain is released,⁴¹ liberating the C-terminal domain. It was therefore interesting to investigate the functional roles of the different domains.In this report, we studied the role of L. major MCA (LmjMCA), using an MCA-deficient strain and overexpressing independently the catalytic and the C-terminal domains. The results confirmed that MCA was not essential to L. major survival. In contrast, LmjMCA processing, releasing its catalytic and C-terminal domains, induced cell death in L. major, whereas the overexpression of Lmjmca gene triggered autophagy after interaction of the C-terminal domain with itself and with other proteins, acting on or upstream of the autophagic protein ATG8.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

RCAD/Ufl1, a Ufm1 E3 ligase,is essential for hematopoietic stem cell function and murine hematopoiesis

The Ufm1 conjugation system is a novel ubiquitin-like modification system, consisting of Ufm1, Uba5 (E1), Ufc1 (E2) and poorly characterized E3 ligase(s). RCAD/Ufl1 (also known as KIAA0776, NLBP and Maxer) was reported to function as a Ufm1 E3 ligase in ufmylation (Ufm1-mediated conjugation) of DDRGK1 and ASC1 proteins. It has also been implicated in estrogen receptor signaling, unfolded protein response (UPR) and neurodegeneration, yet its physiological function remains completely unknown. In this study, we report that RCAD/Ufl1 is essential for embryonic development, hematopoietic stem cell (HSC) survival and erythroid differentiation. Both germ-line and somatic deletion of RCAD/Ufl1 impaired hematopoietic development, resulting in severe anemia, cytopenia and ultimately animal death. Depletion of RCAD/Ufl1 caused elevated endoplasmic reticulum stress and evoked UPR in bone marrow cells. In addition, loss of RCAD/Ufl1 blocked autophagic degradation, increased mitochondrial mass and reactive oxygen species, and led to DNA damage response, p53 activation and enhanced cell death of HSCs. Collectively, our study provides the first genetic evidence for the indispensable role of RCAD/Ufl1 in murine hematopoiesis and development. The finding of RCAD/Ufl1 as a key regulator of cellular stress response sheds a light into the role of a novel protein network including RCAD/Ufl1 and its associated proteins in regulating cellular homeostasis.The Ufm1 (Ubiquitin-fold modifier 1) conjugation system is a novel ubiquitin-like (Ubl) modification system that shares biochemical features with other Ubl systems.¹ Ufm1 modifies its target proteins through a biochemical pathway catalyzed by specific E1 (Uba5), E2 (Ufc1) and E3 enzyme(s) even though the identities of E3 ligases remain mostly elusive. Genetic study from Uba5 knockout (KO) mice has shown that Uba5 is indispensable for embryonic erythropoiesis, highlighting the pivotal role of this novel Ubl system in animal development.² Yet its role in adult erythropoiesis and other developmental processes is largely unexplored and the underlying molecular mechanism remains poorly understood.Regulator of C53 and DDRGK1 (also known as KIAA0776, Ufl1, NLBP and Maxer, referred to as RCAD hereafter) has recently been identified by independent studies as an important regulator of several signaling pathways, including protein ufmylation, NF-κB signaling and unfolded protein response (UPR).^{3, 4, 5, 6, 7, 8, 9} Endogenous RCAD forms a complex with two proteins: C53 (also known as LZAP and Cdk5rap3) ^{5, 6, 10} and DDRGK1 (also designated as C20orf116, Dashurin and UFBP1),^{3, 6, 7, 11} and regulates the stability of its binding partners.^{5, 6} Intriguingly, Tatsumi et al.³ found that Ufl1 (same as RCAD) promoted ufmylation of DDRGK1, suggesting that RCAD may function as an E3 ligase for ufmylation of DDRGK1. In line with its role in ufmylation, knockdown of endogenous RCAD resulted in attenuated ufmylation of endogenous Ufm1 targets.^{7, 8} More recently, Yoo et al.¹² found that Ufl1-mediated ufmylation of ASC1, a nuclear receptor co-activator, played a crucial role in estrogen receptor signaling and breast cancer development. Nonetheless, the in vivo function of RCAD remains completely unknown.In this study, we report the establishment of RCAD KO mouse models. Ablation of RCAD leads to impaired embryogenesis and defective hematopoiesis. Our study provides the first genetic evidence for the indispensable role of this important protein in animal development.