Structural basis of defects in the sacsin HEPN domain responsible for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS)

Sacsin is a 520-kDa protein mutated in the early-onset neurodevelopmental and neurodegenerative disease autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The C terminus of the protein contains an HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain of unknown function. Here, we determined the high-resolution 1.9-Å crystal structure of the HEPN domain from human sacsin. The structure is composed of five parallel α-helices with a large loop of several short helical segments. Two HEPN protomers assemble as a dimer to form a large positively charged cavity at the dimer interface that binds GTP and other nucleotides. The crystal structure reveals that the ARSACS N4549D mutation disrupts dimerization and protein folding. This study provides novel insights into the oligomerization state of sacsin and functions that are lost in mutations that cause ARSACS.     

Attenuation of T Cell Receptor Signaling by Serine Phosphorylation-mediated Lysine 30 Ubiquitination of SLP-76 Protein

SLP-76 (SH2 domain-containing leukocyte protein of 76 kDa) is an adaptor protein that is essential for T cell development and T cell receptor (TCR) signaling activation. Previous studies have identified an important negative feedback regulation of SLP-76 by HPK1 (hematopoietic progenitor kinase 1; MAP4K1)-induced Ser-376 phosphorylation. Ser-376 phosphorylation of SLP-76 mediates 14-3-3 binding, resulting in the attenuation of SLP-76 activation and downstream signaling; however, the underlying mechanism of this action remains unknown. Here, we report that phosphorylated SLP-76 is ubiquitinated and targeted for proteasomal degradation during TCR signaling. SLP-76 ubiquitination is mediated by Ser-376 phosphorylation. Furthermore, Lys-30 is identified as a ubiquitination site of SLP-76. Loss of Lys-30 ubiquitination of SLP-76 results in enhanced anti-CD3 antibody-induced ERK and JNK activation. These results reveal a novel regulation mechanism of SLP-76 by ubiquitination and proteasomal degradation of activated SLP-76, which is mediated by Ser-376 phosphorylation, leading to down-regulation of TCR signaling.     

A bacterial cytotoxin identifies the RhoA exchange factor Net1 as a key effector in the response to DNA damage

Background

Exposure of adherent cells to DNA damaging agents, such as the bacterial cytolethal distending toxin (CDT) or ionizing radiations (IR), activates the small GTPase RhoA, which promotes the formation of actin stress fibers and delays cell death. The signalling intermediates that regulate RhoA activation and promote cell survival are unknown.

Principal Findings

We demonstrate that the nuclear RhoA-specific Guanine nucleotide Exchange Factor (GEF) Net1 becomes dephosphorylated at a critical inhibitory site in cells exposed to CDT or IR. Expression of a dominant negative Net1 or Net1 knock down by iRNA prevented RhoA activation, inhibited the formation of stress fibers, and enhanced cell death, indicating that Net1 activation is required for this RhoA-mediated responses to genotoxic stress. The Net1 and RhoA-dependent signals involved activation of the Mitogen-Activated Protein Kinase p38 and its downstream target MAPK-activated protein kinase 2.

Significance

Our data highlight the importance of Net1 in controlling RhoA and p38 MAPK mediated cell survival in cells exposed to DNA damaging agents and illustrate a molecular pathway whereby chronic exposure to a bacterial toxin may promote genomic instability.     

Lipid Droplet Protein LID-1 Mediates ATGL-1-Dependent Lipolysis during Fasting in Caenorhabditis elegans

Lipolysis is a delicate process involving complex signaling cascades and sequential enzymatic activations. In Caenorhabditis elegans, fasting induces various physiological changes, including a dramatic decrease in lipid contents through lipolysis. Interestingly, C. elegans lacks perilipin family genes which play a crucial role in the regulation of lipid homeostasis in other species. Here, we demonstrate that in the intestinal cells of C. elegans, a newly identified protein, lipid droplet protein 1 (C25A1.12; LID-1), modulates lipolysis by binding to adipose triglyceride lipase 1 (C05D11.7; ATGL-1) during nutritional deprivation. In fasted worms, lipid droplets were decreased in intestinal cells, whereas suppression of ATGL-1 via RNA interference (RNAi) resulted in retention of stored lipid droplets. Overexpression of ATGL-1 markedly decreased lipid droplets, whereas depletion of LID-1 via RNAi prevented the effect of overexpressed ATGL-1 on lipolysis. In adult worms, short-term fasting increased cyclic AMP (cAMP) levels, which activated protein kinase A (PKA) to stimulate lipolysis via ATGL-1 and LID-1. Moreover, ATGL-1 protein stability and LID-1 binding were augmented by PKA activation, eventually leading to increased lipolysis. These data suggest the importance of the concerted action of lipase and lipid droplet protein in the response to fasting signals via PKA to maintain lipid homeostasis.     

RPM-1 Uses Both Ubiquitin Ligase and Phosphatase-Based Mechanisms to Regulate DLK-1 during Neuronal Development

The Pam/Highwire/RPM-1 (PHR) proteins are key regulators of neuronal development that function in axon extension and guidance, termination of axon outgrowth, and synapse formation. Outside of development, the PHR proteins also regulate axon regeneration and Wallerian degeneration. The PHR proteins function in part by acting as ubiquitin ligases that degrade the Dual Leucine zipper-bearing Kinase (DLK). Here, we show that the Caenorhabditis elegans PHR protein, Regulator of Presynaptic Morphology 1 (RPM-1), also utilizes a phosphatase-based mechanism to regulate DLK-1. Using mass spectrometry, we identified Protein Phosphatase Magnesium/Manganese dependent 2 (PPM-2) as a novel RPM-1 binding protein. Genetic, transgenic, and biochemical studies indicated that PPM-2 functions coordinately with the ubiquitin ligase activity of RPM-1 and the F-box protein FSN-1 to negatively regulate DLK-1. PPM-2 acts on S874 of DLK-1, a residue implicated in regulation of DLK-1 binding to a short, inhibitory isoform of DLK-1 (DLK-1S). Our study demonstrates that PHR proteins function through both phosphatase and ubiquitin ligase mechanisms to inhibit DLK. Thus, PHR proteins are potentially more accurate and sensitive regulators of DLK than originally thought. Our results also highlight an important and expanding role for the PP2C phosphatase family in neuronal development.     

Yu Ping Feng San,an Ancient Chinese Herbal Decoction Containing Astragali Radix,Atractylodis Macrocephalae Rhizoma and Saposhnikoviae Radix,Regulates the Release of Cytokines in Murine Macrophages

Yu Ping Feng San (YPFS), a Chinese herbal decoction, is composed of Astragali Radix (AR; Huangqi), Atractylodis Macrocephalae Rhizoma (AMR; Baizhu) and Saposhnikoviae Radix (SR; Fangfeng) in a weight ratio of 1∶2∶1. Clinically, YPFS has been widely used to regulate immune functions; however, the action mechanism of it is not known. Here, we addressed this issue by providing detail analyses of chemical and biological properties of YPFS. By using rapid resolution liquid chromatography coupled with mass spectrometry, fifteen chemicals deriving from different herbs of YPFS were determined, and which served as a control for the standardization of the herbal extract of YPFS. In general, the amounts of chosen chemical markers were higher in a preparation of YPFS as compared to that of single herb or two-herb compositions. In order to reveal the immune functions of YPFS, the standardized extract was applied onto cultured murine macrophages. The treatment of YPFS stimulated the mRNA and protein expressions of pro-inflammatory cytokines via activation of NF-κB by enhancing IκBα degradation. In contrast, the application of YPFS suppressed the expressions of pro-inflammatory cytokines significantly in the lipopolysaccharide (LPS)-induced chronic inflammation model. In addition, YPFS could up regulate the phagocytic activity in cultured macrophages. These results therefore supported the bi-directional immune-modulatory roles of YPFS in regulating the releases of cytokines from macrophages.     

Ankyrin Repeat Domain Protein 2 and Inhibitor of DNA Binding 3 Cooperatively Inhibit Myoblast Differentiation by Physical Interaction

Ankyrin repeat domain protein 2 (ANKRD2) translocates from the nucleus to the cytoplasm upon myogenic induction. Overexpression of ANKRD2 inhibits C2C12 myoblast differentiation. However, the mechanism by which ANKRD2 inhibits myoblast differentiation is unknown. We demonstrate that the primary myoblasts of mdm (muscular dystrophy with myositis) mice (pMB^mdm) overexpress ANKRD2 and ID3 (inhibitor of DNA binding 3) proteins and are unable to differentiate into myotubes upon myogenic induction. Although suppression of either ANKRD2 or ID3 induces myoblast differentiation in mdm mice, overexpression of ANKRD2 and inhibition of ID3 or vice versa is insufficient to inhibit myoblast differentiation in WT mice. We identified that ANKRD2 and ID3 cooperatively inhibit myoblast differentiation by physical interaction. Interestingly, although MyoD activates the Ankrd2 promoter in the skeletal muscles of wild-type mice, SREBP-1 (sterol regulatory element binding protein-1) activates the same promoter in the skeletal muscles of mdm mice, suggesting the differential regulation of Ankrd2. Overall, we uncovered a novel pathway in which SREBP-1/ANKRD2/ID3 activation inhibits myoblast differentiation, and we propose that this pathway acts as a critical determinant of the skeletal muscle developmental program.     

Human ISCA1 Interacts with IOP1/NARFL and Functions in Both Cytosolic and Mitochondrial Iron-Sulfur Protein Biogenesis

Iron-sulfur proteins play an essential role in many biologic processes. Hence, understanding their assembly is an important goal. In Escherichia coli, the protein IscA is a product of the isc (iron-sulfur cluster) operon and functions in the iron-sulfur cluster assembly pathway in this organism. IscA is conserved in evolution, but its function in mammalian cells is not known. Here, we provide evidence for a role for a human homologue of IscA, named IscA1, in iron-sulfur protein biogenesis. We observe that small interfering RNA knockdown of IscA1 in HeLa cells leads to decreased activity of two mitochondrial iron-sulfur enzymes, succinate dehydrogenase and mitochondrial aconitase, as well as a cytosolic iron-sulfur enzyme, cytosolic aconitase. IscA1 is observed both in cytosolic and mitochondrial fractions. We find that IscA1 interacts with IOP1 (iron-only hydrogenase-like protein 1)/NARFL (nuclear prelamin A recognition factor-like), a cytosolic protein that plays a role in the cytosolic iron-sulfur protein assembly pathway. We therefore propose that human IscA1 plays an important role in both mitochondrial and cytosolic iron-sulfur cluster biogenesis, and a notable component of the latter is the interaction between IscA1 and IOP1.     

Characterization of Streptokinases from Group A Streptococci Reveals a Strong Functional Relationship That Supports the Coinheritance of Plasminogen-binding M Protein and Cluster 2b Streptokinase

Group A streptococcus (GAS) strains secrete the protein streptokinase (SK), which functions by activating host human plasminogen (hPg) to plasmin (hPm), thus providing a proteolytic framework for invasive GAS strains. The types of SK secreted by GAS have been grouped into two clusters (SK1 and SK2) and one subcluster (SK2a and SK2b). SKs from cluster 1 (SK1) and cluster 2b (SK2b) display significant evolutionary and functional differences, and attempts to relate these properties to GAS skin or pharynx tropism and invasiveness are of great interest. In this study, using four purified SKs from each cluster, new relationships between plasminogen-binding group A streptococcal M (PAM) protein and SK2b have been revealed. All SK1 proteins efficiently activated hPg, whereas all subclass SK2b proteins only weakly activated hPg in the absence of PAM. Surface plasmon resonance studies revealed that the lower affinity of SK2b to hPg served as the basis for the attenuated activation of hPg by SK2b. Binding of hPg to either human fibrinogen (hFg) or PAM greatly enhanced activation of hPg by SK2b but minimally influenced the already effective activation of hPg by SK1. Activation of hPg in the presence of GAS cells containing PAM demonstrated that PAM is the only factor on the surface of SK2b-expressing cells that enabled the direct activation of hPg by SK2b. As the binding of hPg to PAM is necessary for hPg activation by SK2b, this dependence explains the coinherant relationship between PAM and SK2b and the ability of these particular strains to generate the proteolytic activity that disrupts the innate barriers that limit invasiveness.     

A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase

Ribosome biogenesis in yeast requires 75 small nucleolar RNAs (snoRNAs) and a myriad of cofactors for processing, modification, and folding of the ribosomal RNAs (rRNAs). For the 19 RNA helicases implicated in ribosome synthesis, their sites of action and molecular functions have largely remained unknown. Here, we have used UV cross-linking and analysis of cDNA (CRAC) to reveal the pre-rRNA binding sites of the RNA helicase Rok1, which is involved in early small subunit biogenesis. Several contact sites were identified in the 18S rRNA sequence, which interestingly all cluster in the “foot” region of the small ribosomal subunit. These include a major binding site in the eukaryotic expansion segment ES6, where Rok1 is required for release of the snR30 snoRNA. Rok1 directly contacts snR30 and other snoRNAs required for pre-rRNA processing. Using cross-linking, ligation and sequencing of hybrids (CLASH) we identified several novel pre-rRNA base-pairing sites for the snoRNAs snR30, snR10, U3, and U14, which cluster in the expansion segments of the 18S rRNA. Our data suggest that these snoRNAs bridge interactions between the expansion segments, thereby forming an extensive interaction network that likely promotes pre-rRNA maturation and folding in early pre-ribosomal complexes and establishes long-range rRNA interactions during ribosome synthesis.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.     

Regulation of the Temperature-dependent Activation of Transient Receptor Potential Vanilloid 1 (TRPV1) by Phospholipids in Planar Lipid Bilayers

TRPV1 (transient receptor potential vanilloid 1) proteins are heat-activated nonselective cation channels. TRPV1 channels are polymodal in their function and exhibit multifaceted regulation with various molecular compounds. In this regard, phosphoinositides, particularly phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate, are important channel regulators. However, their effects on TRPV1 channel activity have not been conclusively determined. To characterize temperature-induced activation of TRPV1 in the presence of different phospholipids, we purified the TRPV1 protein from HEK-293 cells and incorporated it into planar lipid bilayers. In the presence of 2.5 μm phosphatidylinositol 4,5-bisphosphate, TRPV1 channels demonstrated rapid activation at 33–39 °C and achieved full channel opening at 42 °C. At this temperature range, TRPV1 heat activation exhibited steep temperature dependence (temperature coefficient (Q₁₀) of 18), and the channel openings were accompanied by large changes in entropy and enthalpy, suggesting a substantial conformation change. At a similar temperature range, another phosphoinositide, phosphatidylinositol 4-phosphate, also potentiated heat activation of TRPV1, but with much lower efficiency. Negatively charged phosphatidylglycerol could also induce heat activation of TRPV1 channels, although with a small-conductance state. Our data demonstrate that phospholipids, specifically phosphoinositides, are important regulators of TRPV1 and are required for heat-induced channel activity.     

Therapeutic Benefits of Induced Pluripotent Stem Cells in Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is characterized by progressive increases in vascular resistance and the remodeling of pulmonary arteries. The accumulation of inflammatory cells in the lung and elevated levels of inflammatory cytokines in the bloodstream suggest that inflammation may play a role in PAH. In this study, the benefits of induced pluripotent stem cells (iPSCs) and iPSC-conditioned medium (iPSC CM) were explored in monocrotaline (MCT)-induced PAH rats. We demonstrated that both iPSCs and iPSC CM significantly reduced the right ventricular systolic pressure and ameliorated the hypertrophy of the right ventricle in MCT-induced PAH rats in models of both disease prevention and disease reversal. In the prevention of MCT-induced PAH, iPSC-based therapy led to the decreased accumulation of inflammatory cells and down-regulated the expression of the IL-1β, IL-6, IL-12α, IL-12β, IL-23 and IFNγ genes in lung specimens, which implied that iPSC-based therapy may be involved in the regulation of inflammation. NF-κB signaling is essential to the inflammatory cascade, which is activated via the phosphorylation of the NF-κB molecule. Using the chemical inhibitor specifically blocked the phosphorylation of NF-κB, and in vitro assays of cultured human M1 macrophages implied that the anti-inflammation effect of iPSC-based therapy may contribute to the disturbance of NF-κB activation. Here, we showed that iPSC-based therapy could restore the hemodynamic function of right ventricle with benefits for preventing the ongoing inflammation in the lungs of MCT-induced PAH rats by regulating NF-κB phosphorylation.     

Structure/Function Analysis of PARP-1 in Oxidative and Nitrosative Stress-Induced Monomeric ADPR Formation

Poly adenosine diphosphate-ribose polymerase-1 (PARP-1) is a multifunctional enzyme that is involved in two major cellular responses to oxidative and nitrosative (O/N) stress: detection and response to DNA damage via formation of protein-bound poly adenosine diphosphate-ribose (PAR), and formation of the soluble 2^nd messenger monomeric adenosine diphosphate-ribose (mADPR). Previous studies have delineated specific roles for several of PARP-1′s structural domains in the context of its involvement in a DNA damage response. However, little is known about the relationship between the mechanisms through which PARP-1 participates in DNA damage detection/response and those involved in the generation of monomeric ADPR. To better understand the relationship between these events, we undertook a structure/function analysis of PARP-1 via reconstitution of PARP-1 deficient DT40 cells with PARP-1 variants deficient in catalysis, DNA binding, auto-PARylation, and PARP-1′s BRCT protein interaction domain. Analysis of responses of the respective reconstituted cells to a model O/N stressor indicated that PARP-1 catalytic activity, DNA binding, and auto-PARylation are required for PARP-dependent mADPR formation, but that BRCT-mediated interactions are dispensable. As the BRCT domain is required for PARP-dependent recruitment of XRCC1 to sites of DNA damage, these results suggest that DNA repair and monomeric ADPR 2^nd messenger generation are parallel mechanisms through which PARP-1 modulates cellular responses to O/N stress.     

Dysregulated Inflammatory Signaling upon Charcot-Marie-Tooth Type 1C Mutation of SIMPLE Protein

Endosomal trafficking is a key mechanism to modulate signal propagation and cross talk. Ubiquitin adaptors, along with endosomal sorting complex required for transport (ESCRT) complexes, are also integrated to terminate ligand-receptor activation in late endosomes and multivesicular bodies (MVBs). Within these pathways, we recently demonstrated that the protein SIMPLE is a novel player in MVB regulation. SIMPLE is also clinically important and its mutation accounts for the Charcot-Marie-Tooth type 1C (CMT1C) disease. MVB defects of mutation and deletion of SIMPLE, however, are distinct. Here, we show that MVB defects found in mutation but not deletion of SIMPLE lead to impaired turnover and accumulation of ESCRT-0 protein Hrs puncta in late endosomes. We further uncover increased colocalization of ubiquitin ligase TRAF6 and Hrs in late endosomes. Upon stimulation with interkeukin-1 or transforming growth factor β, prolonged activation of p38 kinase/JNK is detected, while nuclear accumulation of NF-κB and phosphorylation of SMAD2 is reduced with CMT1C mutation. The aberrant kinetics we observed in inflammatory signaling may contribute to increased tumor susceptibility and changes in the levels of chemokines/cytokines that result from CMT1C mutation. We propose that altered endosomal trafficking due to malformations of MVBs and subsequent atypical signaling kinetic may account for a toxic gain of function in CMT1C pathogenesis.     

MMS21/HPY2 and SIZ1, Two Arabidopsis SUMO E3 Ligases,Have Distinct Functions in Development

The small ubiquitin related modifier (SUMO)-mediated posttranslational protein modification is widely conserved among eukaryotes. Similar to ubiquitination, SUMO modifications are attached to the substrate protein through three reaction steps by the E1, E2 and E3 enzymes. To date, multiple families of SUMO E3 ligases have been reported in yeast and animals, but only two types of E3 ligases have been identified in Arabidopsis: SAP and Miz 1 (SIZ1) and Methyl Methanesulfonate-Sensitivity protein 21 (MMS21)/HIGH PLOIDY 2 (HPY2), hereafter referred to as HPY2. Both proteins possess characteristic motifs termed Siz/PIAS RING (SP-RING) domains, and these motifs are conserved throughout the plant kingdom. Previous studies have shown that loss-of-function mutations in HPY2 or SIZ1 cause dwarf phenotypes and that the phenotype of siz1-2 is caused by the accumulation of salicylic acid (SA). However, we demonstrate here that the phenotype of hpy2-1 does not depend on SA accumulation. Consistently, the expression of SIZ1 driven by the HPY2 promoter does not complement the hpy2-1 phenotypes, indicating that they are not functional homologs. Lastly, we show that the siz1-2 and hpy2-1 double mutant results in embryonic lethality, supporting the hypothesis that they have non-overlapping roles during embryogenesis. Together, these results suggest that SIZ1 and HPY2 function independently and that their combined SUMOylation is essential for plant development.