Homozygous EPRS1 missense variant causing hypomyelinating leukodystrophy-15 alters variant-distal mRNA m6A site accessibility.

Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N6-methyladenosine (m6A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m6A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m6A sites on target EPRS1 mRNA, or by m6A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m6A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders.

Aryl hydrocarbon receptor restricts axon regeneration of DRG neurons in response to injury.

Injured neurons sense environmental cues to balance neural protection and axon regeneration, but the mechanisms are unclear. Here, we unveil aryl hydrocarbon receptor (AhR), a ligand-activated bHLH-PAS transcription factor, as molecular sensor and key regulator of acute stress response at the expense of axon regeneration. We demonstrate responsiveness of DRG sensory neurons to ligand-mediated AhR signaling, which functions to inhibit axon regeneration. Ahr deletion mimics the conditioning lesion in priming DRG to initiate axonogenesis gene programs; upon peripheral axotomy, Ahr ablation suppresses inflammation and stress signaling while augmenting pro-growth pathways. Moreover, comparative transcriptomics revealed signaling interactions between AhR and HIF-1α, two structurally related bHLH-PAS α units that share the dimerization partner Arnt/HIF-1ß. Functional assays showed that the growth advantage of AhR-deficient DRG neurons requires HIF-1α; but in the absence of Arnt, DRG neurons can still mount a regenerative response. We further unveil a link between bHLH-PAS transcription factors and DNA hydroxymethylation in response to peripheral axotomy, while neuronal single cell RNA-seq analysis revealed a link of the AhR regulon to RNA polymerase III regulation and integrated stress response (ISR). Altogether, AhR activation favors stress coping and inflammation at the expense of axon regeneration; targeting AhR can enhance nerve repair.

Circadian clock regulator Bmal1 gates axon regeneration via Tet3 epigenetics in mouse sensory neurons.

Axon regeneration of dorsal root ganglia (DRG) neurons after peripheral axotomy involves reconfiguration of gene regulatory circuits to establish regenerative gene programs. However, the underlying mechanisms remain unclear. Here, through an unbiased survey, we show that the binding motif of Bmal1, a central transcription factor of the circadian clock, is enriched in differentially hydroxymethylated regions (DhMRs) of mouse DRG after peripheral lesion. By applying conditional deletion of Bmal1 in neurons, in vitro and in vivo neurite outgrowth assays, as well as transcriptomic profiling, we demonstrate that Bmal1 inhibits axon regeneration, in part through a functional link with the epigenetic factor Tet3. Mechanistically, we reveal that Bmal1 acts as a gatekeeper of neuroepigenetic responses to axonal injury by limiting Tet3 expression and restricting 5hmC modifications. Bmal1-regulated genes not only concern axon growth, but also stress responses and energy homeostasis. Furthermore, we uncover an epigenetic rhythm of diurnal oscillation of Tet3 and 5hmC levels in DRG neurons, corresponding to time-of-day effect on axon growth potential. Collectively, our studies demonstrate that targeting Bmal1 enhances axon regeneration.

Macrophages facilitate peripheral nerve regeneration by organizing regeneration tracks through Plexin-B2.

The regeneration of peripheral nerves is guided by regeneration tracks formed through an interplay of many cell types, but the underlying signaling pathways remain unclear. Here, we demonstrate that macrophages are mobilized ahead of Schwann cells in the nerve bridge after transection injury to participate in building regeneration tracks. This requires the function of guidance receptor Plexin-B2, which is robustly up-regulated in infiltrating macrophages in injured nerves. Conditional deletion of Plexin-B2 in myeloid lineage resulted in not only macrophage misalignment but also matrix disarray and Schwann cell disorganization, leading to misguided axons and delayed functional recovery. Plexin-B2 is not required for macrophage recruitment or activation but enables macrophages to steer clear of colliding axons, in particular the growth cones at the tip of regenerating axons, leading to parallel alignment postcollision. Together, our studies unveil a novel reparative function of macrophages and the importance of Plexin-B2-mediated collision-dependent contact avoidance between macrophages and regenerating axons in forming regeneration tracks during peripheral nerve regeneration.

Epigenetic Regulation Of Axon Regeneration and Glial Activation in Injury Responses.

Injury to the nervous system triggers a multicellular response in which epigenetic mechanisms play an important role in regulating cell type-specific transcriptional changes. Here, we summarize recent progress in characterizing neuronal intrinsic and extrinsic chromatin reconfigurations and epigenetic changes triggered by axonal injury that shape neuroplasticity and glial functions. We specifically discuss regeneration-associated transcriptional modules comprised of transcription factors and epigenetic regulators that control axon growth competence. We also review epigenetic regulation of neuroinflammation and astroglial responses that impact neural repair. These advances provide a framework for developing epigenetic strategies to maximize adaptive alterations while minimizing maladaptive stress responses in order to enhance axon regeneration and achieve functional recovery after injury.

Structural control of caspase-generated glutamyl-tRNA synthetase by appended noncatalytic WHEP domains.

Aminoacyl-tRNA synthetases are ubiquitous, evolutionarily conserved enzymes catalyzing the conjugation of amino acids onto cognate tRNAs. During eukaryotic evolution, tRNA synthetases have been the targets of persistent structural modifications. These modifications can be additive, as in the evolutionary acquisition of noncatalytic domains, or subtractive, as in the generation of truncated variants through regulated mechanisms such as proteolytic processing, alternative splicing, or coding region polyadenylation. A unique variant is the human glutamyl-prolyl-tRNA synthetase (EPRS) consisting of two fused synthetases joined by a linker containing three copies of the WHEP domain (termed by its presence in tryptophanyl-, histidyl-, and glutamyl-prolyl-tRNA synthetases). Here, we identify site-selective proteolysis as a mechanism that severs the linkage between the EPRS synthetases in vitro and in vivo Caspase action targeted Asp-929 in the third WHEP domain, thereby separating the two synthetases. Using a neoepitope antibody directed against the newly exposed C terminus, we demonstrate EPRS cleavage at Asp-929 in vitro and in vivo Biochemical and biophysical characterizations of the N-terminally generated EPRS proteoform containing the glutamyl-tRNA synthetase and most of the linker, including two WHEP domains, combined with structural analysis by small-angle neutron scattering, revealed a role for the WHEP domains in modulating conformations of the catalytic core and GSH-S-transferase-C-terminal-like (GST-C) domain. WHEP-driven conformational rearrangement altered GST-C domain interactions and conferred distinct oligomeric states in solution. Collectively, our results reveal long-range conformational changes imposed by the WHEP domains and illustrate how noncatalytic domains can modulate the global structure of tRNA synthetases in complex eukaryotic systems.

Condensin II and GAIT complexes cooperate to restrict LINE-1 retrotransposition in epithelial cells.

LINE-1 (L1) retrotransposons can mobilize (retrotranspose) within the human genome, and mutagenic de novo L1 insertions can lead to human diseases, including cancers. As a result, cells are actively engaged in preventing L1 retrotransposition. This work reveals that the human Condensin II complex restricts L1 retrotransposition in both non-transformed and transformed cell lines through inhibition of L1 transcription and translation. Condensin II subunits, CAP-D3 and CAP-H2, interact with members of the Gamma-Interferon Activated Inhibitor of Translation (GAIT) complex including the glutamyl-prolyl-tRNA synthetase (EPRS), the ribosomal protein L13a, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and NS1 associated protein 1 (NSAP1). GAIT has been shown to inhibit translation of mRNAs encoding inflammatory proteins in myeloid cells by preventing the binding of the translation initiation complex, in response to Interferon gamma (IFN-γ). Excitingly, our data show that Condensin II promotes complexation of GAIT subunits. Furthermore, RNA-Immunoprecipitation experiments in epithelial cells demonstrate that Condensin II and GAIT subunits associate with L1 RNA in a co-dependent manner, independent of IFN-γ. These findings suggest that cooperation between the Condensin II and GAIT complexes may facilitate a novel mechanism of L1 repression, thus contributing to the maintenance of genome stability in somatic cells.

EPRS is a critical mTORC1-S6K1 effector that influences adiposity in mice.

Metabolic pathways that contribute to adiposity and ageing are activated by the mammalian target of rapamycin complex 1 (mTORC1) and p70 ribosomal protein S6 kinase 1 (S6K1) axis. However, known mTORC1-S6K1 targets do not account for observed loss-of-function phenotypes, suggesting that there are additional downstream effectors of this pathway. Here we identify glutamyl-prolyl-tRNA synthetase (EPRS) as an mTORC1-S6K1 target that contributes to adiposity and ageing. Phosphorylation of EPRS at Ser999 by mTORC1-S6K1 induces its release from the aminoacyl tRNA multisynthetase complex, which is required for execution of noncanonical functions of EPRS beyond protein synthesis. To investigate the physiological function of EPRS phosphorylation, we generated Eprs knock-in mice bearing phospho-deficient Ser999-to-Ala (S999A) and phospho-mimetic (S999D) mutations. Homozygous S999A mice exhibited low body weight, reduced adipose tissue mass, and increased lifespan, similar to S6K1-deficient mice and mice with adipocyte-specific deficiency of raptor, an mTORC1 constituent. Substitution of the EprsS999D allele in S6K1-deficient mice normalized body mass and adiposity, indicating that EPRS phosphorylation mediates S6K1-dependent metabolic responses. In adipocytes, insulin stimulated S6K1-dependent EPRS phosphorylation and release from the multisynthetase complex. Interaction screening revealed that phospho-EPRS binds SLC27A1 (that is, fatty acid transport protein 1, FATP1), inducing its translocation to the plasma membrane and long-chain fatty acid uptake. Thus, EPRS and FATP1 are terminal mTORC1-S6K1 axis effectors that are critical for metabolic phenotypes.

Experimental approaches for investigation of aminoacyl tRNA synthetase phosphorylation.

Phosphorylation of many aminoacyl tRNA synthetases (AARSs) has been recognized for decades, but the contribution of post-translational modification to their primary role in tRNA charging and decryption of genetic code remains unclear. In contrast, phosphorylation is essential for performance of diverse noncanonical functions of AARSs unrelated to protein synthesis. Phosphorylation of glutamyl-prolyl tRNA synthetase (EPRS) has been investigated extensively in our laboratory for more than a decade, and has served as an archetype for studies of other AARSs. EPRS is a constituent of the IFN-γ-activated inhibitor of translation (GAIT) complex that directs transcript-selective translational control in myeloid cells. Stimulus-dependent phosphorylation of EPRS is essential for its release from the parental multi-aminoacyl tRNA synthetase complex (MSC), for binding to other GAIT complex proteins, and for regulating the binding to target mRNAs. Importantly, phosphorylation is the common driving force for the context- and stimulus-dependent release, and non-canonical activity, of other AARSs residing in the MSC, for example, lysyl tRNA synthetase (KARS). Here, we describe the concepts and experimental methodologies we have used to investigate the influence of phosphorylation on the structure and function of EPRS. We suggest that application of these approaches will help to identify new functional phosphorylation event(s) in other AARSs and elucidate their possible roles in noncanonical activities.

Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring.

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring.

Identification of Caspase-6-mediated processing of the valosin containing protein (p97) in Alzheimer's disease: a novel link to dysfunction in ubiquitin proteasome system-mediated protein degradation.

The valosin-containing protein (p97) is a ubiquitin-dependent ATPase that plays central roles in ubiquitin proteasome system (UPS)-mediated protein degradation pathways. p97 has been recently identified as a putative substrate of active Caspase-6 (Casp6) in primary human neurons. Since Casp6 is activated in mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients' brains, the targeting of p97 by Casp6 may represent an important step that leads to UPS impairment in AD. Here, we show that p97 is a Casp6 substrate in vitro and in vivo. Casp6 cleavage of recombinant p97 generated two N-terminal fragments of 28 and 20 kDa, which were not generated by the other two effector caspases, Caspase-3 and Caspase-7. ATP binding to the D1 ATPase ring of p97 reduced the susceptibility of the N-domain to caspase-mediated proteolysis. Mass spectrometric analysis identified VAPD(179) as a Casp6 cleavage site within p97's N-domain. An anti-neoepitope serum immunohistochemically detected p97 cleaved at VAPD(179) in the cytoplasm of the cell soma and neurites of hippocampal neurons in MCI and AD. Overexpression of p97 (1-179) fragment, representing p97 cleaved at D179, impaired the degradation of model substrates in the ubiquitin-fusion degradation and the N-end rule pathways, and destabilized endogenous p97. Collectively, these results show that p97 is cleaved by Casp6 in AD and suggest p97 cleavage as an important mechanism for UPS impairment.

Hereditary inclusion body myopathy-linked p97/VCP mutations in the NH2 domain and the D1 ring modulate p97/VCP ATPase activity and D2 ring conformation.

Hereditary inclusion body myopathy associated with early-onset Paget disease of bone and frontotemporal dementia (hIBMPFTD) is a degenerative disorder caused by single substitutions in highly conserved residues of p97/VCP. All mutations identified thus far cluster within the NH(2) domain or the D1 ring, which are both required for communicating conformational changes to adaptor protein complexes. In this study, biochemical approaches were used to identify the consequences of the mutations R155P and A232E on p97/VCP structure. Assessment of p97/VCP oligomerization revealed that p97(R155P) and p97(A232E) formed hexameric ring-shaped structures of approximately 600 kDa. p97(R155P) and p97(A232E) exhibited an approximately 3-fold increase in ATPase activity compared to wild-type p97 (p97(WT)) and displayed increased sensitivity to heat-induced upregulation of ATPase activity. Protein fluorescence analysis provided evidence for conformational differences in the D2 rings of both hIBMPFTD mutants. Furthermore, both mutations increased the proteolytic susceptibility of the D2 ring. The solution structures of all p97/VCP proteins revealed a didispersed distribution of a predominant hexameric population and a minor population of large-diameter complexes. ATP binding significantly increased the abundance of large-diameter complexes for p97(R155P) and p97(A232E), but not p97(WT) or the ATP-binding mutant p97(K524A). Therefore, we propose that hIBMPFTD p97/VCP mutants p97(R155P) and p97(A232E) possess structural defects that may compromise the mechanism of p97/VCP activity within large multiprotein complexes.

GTPase-mediated regulation of the unfolded protein response in Caenorhabditis elegans is dependent on the AAA+ ATPase CDC-48.

When endoplasmic reticulum (ER) homeostasis is perturbed, an adaptive mechanism is triggered and named the unfolded protein response (UPR). Thus far, three known UPR signaling branches (IRE-1, PERK, and ATF-6) mediate the reestablishment of ER functions but can also lead to apoptosis if ER stress is not alleviated. However, the understanding of the molecular mechanisms integrating the UPR to other ER functions, such as membrane traffic or endomembrane signaling, remains incomplete. We consequently sought to identify new regulators of UPR-dependent transcriptional mechanisms and focused on a family of proteins known to mediate, among other, ER-related functions: the small GTP-binding proteins of the RAS superfamily. To this end, we used transgenic UPR reporter Caenorhabditis elegans strains as a model to specifically silence small-GTPase expression. We show that the Rho subfamily member CRP-1 is an essential component of UPR-induced transcriptional events through its physical and genetic interactions with the AAA+ ATPase CDC-48. In addition, we describe a novel signaling module involving CRP-1 and CDC-48 which may directly link the UPR to DNA remodeling and transcription control.

Requirements for the selective degradation of CD4 receptor molecules by the human immunodeficiency virus type 1 Vpu protein in the endoplasmic reticulum.

BACKGROUND: HIV-1 Vpu targets newly synthesized CD4 receptor for rapid degradation by a process reminiscent of endoplasmic reticulum (ER)-associated protein degradation (ERAD). Vpu is thought to act as an adaptor protein, connecting CD4 to the ubiquitin (Ub)-proteasome degradative system through an interaction with beta-TrCP, a component of the SCFbeta-TrCP E3 Ub ligase complex. RESULTS: Here, we provide direct evidence indicating that Vpu promotes trans-ubiquitination of CD4 through recruitment of SCFbeta-TrCP in human cells. To examine whether Ub conjugation occurs on the cytosolic tail of CD4, we substituted all four Ub acceptor lysine residues for arginines. Replacement of cytosolic lysine residues reduced but did not prevent Vpu-mediated CD4 degradation and ubiquitination, suggesting that Vpu-mediated CD4 degradation is not entirely dependent on the ubiquitination of cytosolic lysines and as such might also involve ubiquitination of other sites. Cell fractionation studies revealed that Vpu enhanced the levels of ubiquitinated forms of CD4 detected in association with not only the ER membrane but also the cytosol. Interestingly, significant amounts of membrane-associated ubiquitinated CD4 appeared to be fully dislocated since they could be recovered following sodium carbonate salt treatment. Finally, expression of a transdominant negative mutant of the AAA ATPase Cdc48/p97 involved in the extraction of ERAD substrates from the ER membrane inhibited Vpu-mediated CD4 degradation. CONCLUSION: Taken together, these results are consistent with a model whereby HIV-1 Vpu targets CD4 for degradation by an ERAD-like process involving most likely poly-ubiquitination of the CD4 cytosolic tail by SCFbeta-TrCP prior to dislocation of receptor molecules across the ER membrane by a process that depends on the AAA ATPase Cdc48/p97.

p97: The cell's molecular purgatory?

The multifunctional AAA-ATPase p97/VCP is one of the most extensively studied members of this protein family, yet it presents the field with many perplexing questions surrounding its mechanism of substrate engagement and processing. Recent discoveries have unmasked a new purgatorial identity for this molecule in the ubiquitin-proteasome pathway, specifically its role in linking ubiquitylated substrates with competing ubiquitin conjugation and deconjugation machineries. Furthermore, biochemical studies surprisingly identify the C-terminal D2 ring as essential for substrate interaction, thus bringing p97 one step closer to its prokaryotic AAA protease relatives.

p38 MAP kinase signaling is necessary for rat chondrosarcoma cell proliferation.

Chondrosarcomas represent the second most frequent class of primary skeletal malignancies. This tumor type is highly resistant to radiation therapy and currently available chemotherapies, thereby limiting treatment choice to surgical resection. Identifying the mechanisms responsible for chondrosarcoma cell proliferation is therefore crucial for the development of new treatment strategies. Here, we demonstrate a significant reduction in rat chondrosarcoma cell proliferation following treatment with pharmacological inhibitors (SB202190 and PD169316) of p38 mitogen-activated protein (MAP) kinases. In an attempt to dissect possible mechanisms, we investigated the effect of p38 inhibition on promoter activity of cell-cycle genes. Surprisingly, p38 inhibition resulted in upregulation of the activities of all three D-type cyclin promoters. In addition, p38 inhibitors induced increased transcription of the cell-cycle inhibitor p21(waf1/cip1). As expected, promoter activity of the cyclin A gene, which lies downstream of D-type cyclins and p21 in cell-cycle progression, was strongly reduced by p38 inhibitors. These effects were independent of a cyclic AMP response element and conferred by the proximal 150 nucleotides of the cyclin A promoter. Decreased transcription was accompanied by greatly reduced cyclin A protein levels upon p38 inhibition. These observations indicate complex regulation of chondrosarcoma cell-cycle progression by p38 signaling, and suggest that components of p38 MAP kinase pathways may be effective targets in the treatment of these tumors.