Sensory neuron–derived NaV1.7 contributes to dorsal horn neuron excitability

Expression of the voltage-gated sodium channel NaV1.7 in sensory neurons is required for pain sensation. We examined the role of NaV1.7 in the dorsal horn of the spinal cord using an epitope-tagged NaV1.7 knock-in mouse. Immuno–electron microscopy showed the presence of NaV1.7 in dendrites of superficial dorsal horn neurons, despite the absence of mRNA. Rhizotomy of L5 afferent nerves lowered the levels of NaV1.7 in the dorsal horn. Peripheral nervous system–specific NaV1.7 null mutant mice showed central deficits, with lamina II dorsal horn tonic firing neurons more than halved and single spiking neurons more than doubled. NaV1.7 blocker PF05089771 diminished excitability in dorsal horn neurons but had no effect on NaV1.7 null mutant mice. These data demonstrate an unsuspected functional role of primary afferent neuron-generated NaV1.7 in dorsal horn neurons and an expression pattern that would not be predicted by transcriptomic analysis

Tools for analysis and conditional deletion of subsets of sensory neurons [version 1; peer review: 4 approved]

Background: Somatosensation depends on primary sensory neurons of the trigeminal and dorsal root ganglia (DRG). Transcriptional profiling of mouse DRG sensory neurons has defined at least 18 distinct neuronal cell types. Using an advillin promoter, we have generated a transgenic mouse line that only expresses diphtheria toxin A (DTA) in sensory neurons in the presence of Cre recombinase. This has allowed us to ablate specific neuronal subsets within the DRG using a range of established and novel Cre lines that encompass all sets of sensory neurons. // Methods: A floxed-tdTomato-stop-DTA bacterial artificial chromosome (BAC) transgenic reporter line (AdvDTA) under the control of the mouse advillin DRG promoter was generated. The line was first validated using a Nav1.8Cre and then crossed to CGRPCreER (Calca), ThCreERT2, Tmem45bCre, Tmem233Cre, Ntng1Cre and TrkBCreER (Ntrk2) lines. Pain behavioural assays included Hargreaves’, hot plate, Randall-Selitto, cold plantar, partial sciatic nerve ligation and formalin tests. // Results: Motor activity, as assessed by the rotarod test, was normal for all lines tested. Noxious mechanosensation was significantly reduced when either Nav1.8 positive neurons or Tmem45b positive neurons were ablated whilst acute heat pain was unaffected. In contrast, noxious mechanosensation was normal following ablation of CGRP-positive neurons but acute heat pain thresholds were significantly elevated and a reduction in nocifensive responses was observed in the second phase of the formalin test. Ablation of TrkB-positive neurons led to significant deficits in mechanical hypersensitivity in the partial sciatic nerve ligation neuropathic pain model. // Conclusions: Ablation of specific DRG neuronal subsets using the AdvDTA line will be a useful resource for further functional characterization of somatosensory processing, neuro-immune interactions and chronic pain disorders

P5A-Type ATPase Cta4p Is Essential for Ca2+ Transport in the Endoplasmic Reticulum of Schizosaccharomyces pombe

This study establishes the role of P5A-type Cta4 ATPase in Ca2+ sequestration in the endoplasmic reticulum by detecting an ATP-dependent, vanadate-sensitive and FCCP insensitive 45Ca2+-transport in fission yeast membranes isolated by cellular fractionation. Specifically, the Ca2+-ATPase transport activity was decreased in ER membranes isolated from cells lacking a cta4+ gene. Furthermore, a disruption of cta4+ resulted in 6-fold increase of intracellular Ca2+ levels, sensitivity towards accumulation of misfolded proteins in ER and ER stress, stimulation of the calcineurin phosphatase activity and vacuolar Ca2+ pumping. These data provide compelling biochemical evidence for a P5A-type Cta4 ATPase as an essential component of Ca2+ transport system and signaling network which regulate, in conjunction with calcineurin, the ER functionality in fission yeast

Proteolytic cleavage of p53 mutants in response to mismatched DNA

Interaction of p53 with mismatched DNA induces proteolytic cleavage with release of a 35-kDa protein fragment from the p53–DNA complexes. The 35-kDa cleavage product is activated for specific biochemical function(s) and may play a role in the cellular response to DNA damage (Molinari et al (1996) Oncogene13: 2077–2086; Okorokov et al (1997) EMBO J16: 6008–6017). In the present study we have asked if mutants of p53 retain the ability to undergo similar proteolytic cleavage, and compared sequence-specific ‘DNA contact’ with ‘structural’ mutants commonly found in human cancer. In addition, a series of phosphorylation site mutants were generated to investigate the possible effects of phosphorylation/dephosphorylation on the proteolytic cleavage of p53. All mutants tested bound to a mismatched DNA target in vitro. Moreover, studies in vitro and in vivo indicate that p53 mutants with intact conformational structure (as determined by immunoreactivity with PAb246 and PAb1620) retain the ability to undergo proteolytic cleavage similar, if not identical, to the wild-type p53 protein. Our results suggest that the capacity for p53 to bind mismatched DNA is independent of structural conformation of the central core domain. Proteolytic cleavage, however, is crucially dependent upon a wild-type conformation of the protein. © 1999 Cancer Research Campaig

Cooperativity Dominates the Genomic Organization of p53-Response Elements: A Mechanistic View

p53-response elements (p53-REs) are organized as two repeats of a palindromic DNA segment spaced by 0 to 20 base pairs (bp). Several experiments indicate that in the vast majority of the human p53-REs there are no spacers between the two repeats; those with spacers, particularly with sizes beyond two nucleotides, are rare. This raises the question of what it indicates about the factors determining the p53-RE genomic organization. Clearly, given the double helical DNA conformation, the orientation of two p53 core domain dimers with respect to each other will vary depending on the spacer size: a small spacer of 0 to 2 bps will lead to the closest p53 dimer-dimer orientation; a 10-bp spacer will locate the p53 dimers on the same DNA face but necessitate DNA looping; while a 5-bp spacer will position the p53 dimers on opposite DNA faces. Here, via conformational analysis we show that when there are 0–2 bp spacers, p53-DNA binding is cooperative; however, cooperativity is greatly diminished when there are spacers with sizes beyond 2 bp. Cooperative binding is broadly recognized to be crucial for biological processes, including transcriptional regulation. Our results clearly indicate that cooperativity of the p53-DNA association dominates the genomic organization of the p53-REs, raising questions of the structural organization and functional roles of p53-REs with larger spacers. We further propose that a dynamic landscape scenario of p53 and p53-REs can better explain the selectivity of the degenerate p53-REs. Our conclusions bear on the evolutionary preference of the p53-RE organization and as such, are expected to have broad implications to other multimeric transcription factor response element organization

Tumor Suppressor RASSF1A Promoter: p53 Binding and Methylation

Oncogenes and tumor suppressors work in concert to regulate cell growth or death, which is a pair of antagonist factors for regulation of tumorigenesis. Here we show promoter characteristic of tumor suppressor RASSF1A, which revealed a p53 binding site in the distal and a GC-rich region in the proximal promoter region of RASSF1A, in despite of TATA box-less. The GC-rich region, which is ∼300 bp upstream from the RASSF1A ATG, showed the strongest promoter activity in an assay of RASSF1A-driving GFP expression. Methylation analysis of the CpG island showed that 78.57% of the GC sties were methylated in testis tumor samples compared with methylation-less in normal testis. Hypermethylation of the GC-rich region is associated with RASSF1A silencing in human testis tumors. In addition, electrophoretic mobility shift assay indicated that p53 protein bound to the RASSF1A promoter. Further chromatin immunoprecipitation confirmed p53 binding to the RASSF1A. Moreover, p53 binding to the promoter down-regulated RASSF1A expression. These results suggest that p53 protein specifically binds to the RASSF1A promoter and inhibits its expression. Our results provide new insight into the mechanism of action of tumor suppressors and may be a starting point for development of new approaches to cancer treatment

p53 Amino-Terminus Region (1–125) Stabilizes and Restores Heat Denatured p53 Wild Phenotype

BACKGROUND:The intrinsically disordered N-ter domain (NTD) of p53 encompasses approximately hundred amino acids that contain a transactivation domain (1-73) and a proline-rich domain (64-92) and is responsible for transactivation function and apoptosis. It also possesses an auto-inhibitory function as its removal results in remarkable reduction in dissociation of p53 from DNA. PRINCIPAL FINDINGS/METHODOLOGY:In this report, we have discovered that p53-NTD spanning amino acid residues 1-125 (NTD125) interacted with WT p53 and stabilized its wild type conformation under physiological and elevated temperatures, both in vitro and in cellular systems. NTD125 prevented irreversible thermal aggregation of heat denatured p53, enhanced p21-5'-DBS binding and further restored DBS binding activity of heat-denatured p53, in vitro, in a dose-dependent manner. In vivo ELISA and immunoprecipitation analysis of NTD125-transfected cells revealed that NTD125 shifted equilibrium from p53 mutant to wild type under heat stress conditions. Further, NTD125 initiated nuclear translocation of cytoplasmic p53 in transcriptionally active state in order to activate p53 downstream genes such as p21, Bax, PUMA, Noxa and SUMO. CONCLUSION/SIGNIFICANCE:Here, we showed that a novel chaperone-like activity resides in p53-N-ter region. This study might have significance in understanding the role of p53-NTD in p53 stabilization, conformational activation and apoptosis under heat-stress conditions

Lysine120 Interactions with p53 Response Elements can Allosterically Direct p53 Organization

p53 can serve as a paradigm in studies aiming to figure out how allosteric perturbations in transcription factors (TFs) triggered by small changes in DNA response element (RE) sequences, can spell selectivity in co-factor recruitment. p53-REs are 20-base pair (bp) DNA segments specifying diverse functions. They may be located near the transcription start sites or thousands of bps away in the genome. Their number has been estimated to be in the thousands, and they all share a common motif. A key question is then how does the p53 protein recognize a particular p53-RE sequence among all the similar ones? Here, representative p53-REs regulating diverse functions including cell cycle arrest, DNA repair, and apoptosis were simulated in explicit solvent. Among the major interactions between p53 and its REs involving Lys120, Arg280 and Arg248, the bps interacting with Lys120 vary while the interacting partners of other residues are less so. We observe that each p53-RE quarter site sequence has a unique pattern of interactions with p53 Lys120. The allosteric, DNA sequence-induced conformational and dynamic changes of the altered Lys120 interactions are amplified by the perturbation of other p53-DNA interactions. The combined subtle RE sequence-specific allosteric effects propagate in the p53 and in the DNA. The resulting amplified allosteric effects far away are reflected in changes in the overall p53 organization and in the p53 surface topology and residue fluctuations which play key roles in selective co-factor recruitment. As such, these observations suggest how similar p53-RE sequences can spell the preferred co-factor binding, which is the key to the selective gene transactivation and consequently different functional effects

A novel human pain insensitivity disorder caused by a point mutation in ZFHX2

Chronic pain is a major global public health issue causing a severe impact on both the quality of life for sufferers and the wider economy. Despite the significant clinical burden, little progress has been made in terms of therapeutic development. A uniquepowerful approach to identifying new human-validated analgesic drug targets is to study rare families with inherited pain insensitivity. Here we have analysed an otherwise normal family where six affected individuals display a pain insensitive phenotype that is characterized by hyposensitivity to noxious heat and painless bone fractures. This autosomal dominant disorder is found in three generations and is not associated with a peripheral neuropathy. A novel point mutation in ZFHX2, encoding a putative transcription factor expressed in small diameter sensory neurons, was identified by whole exome sequencing that segregates with the pain insensitivity. The mutation is predicted to change an evolutionarily highly conserved arginine residue 1913 to a lysine within a homeodomain. BAC transgenic mice bearing the orthologous murine p.R1907K mutation, as well as Zfhx2 null mutant mice, have significant deficits in pain sensitivity. Gene expression analyses in dorsal root ganglia from mutant and wild-type mice show altered expression of genes implicated in peripheral pain mechanisms. The ZFHX2 variant and downstream regulated genes associated with a human paininsensitive phenotype are therefore potential novel targets for the development of new analgesic drugs