An eccentric calpain, CAPN3/p94/calpain-3

AbstractCalpains are Ca2+-regulated proteolytic enzymes that are involved in a variety of biological phenomena. Calpains process substrates by limited proteolysis to modulate various protein functions in the cell, and are thus called “modulator proteases.” CAPN3, previously called p94 or calpain-3, has unique features that are not found in any of the other 14 human calpains, or even in other proteases.For instance, CAPN3 undergoes extremely rapid and exhaustive autodegradation. CAPN3 is also the first (and so far, the only) intracellular enzyme found to depend on Na+ for its activation. CAPN3 has both proteolytic and non-proteolytic functions. It has the interesting distinction of being the only protease, other than a few virus proteases, with the ability to regain protease function after its autolytic dissociation; this occurs through a process known as intermolecular complementation (iMOC). Gene mutations causing CAPN3 defects are responsible for limb-girdle muscular dystrophy type 2A (LGMD2A).Unusual characteristics of CAPN3 have fascinated researchers, but have also hampered conventional biochemical analysis. In this review, we describe significant findings about CAPN3 from its discovery to the present, and suggest promising avenues for future CAPN3 research

Clinical and genetic aspects of mild hypophosphatasia in Japanese patients

Background: Hypophosphatasia (HPP) is a rare inborn error of metabolism that results from a dysfunctional tissue non-specific alkaline phosphatase enzyme (TNSALP). Although genotype-phenotype correlations have been described in HPP patients, only sparse information is currently available on the genetics of mild type HPP. Methods: We investigated 5 Japanese patients from 3 families with mild HPP (patients 1 and 2 are siblings; patient 4 is a daughter of patient 5) who were referred to Fujita Health University due to the premature loss of deciduous teeth. Physical and dental examinations, and blood, urine and bone density tests were conducted. Genetic analysis of the ALPL gene was performed in all patients with their informed consent. Results: After a detailed interview and examination, we found characteristic symptoms of HPP in some of the study cases. Mobile teeth or the loss of permanent teeth were observed in 2 patients, and 3 out of 5 patients had a history of asthma. The serum ALP levels of all patients were 30% below the lower limit of the age equivalent normal range. ALPL gene analysis revealed compound heterozygous mutations, including Ile395Val and Leu520Argfs in family 1, Val95Met and Gly491Arg in family 2, and a dominant missense mutation (Gly456Arg) in family 3. The 3D-modeling of human TNSALP revealed three mutations (Val95Met, Ile395Val and Gly456Arg) at the homodimer interface. Severe collisions between the side chains were predicted for the Gly456Arg variant. Discussion: One of the characteristic findings of this present study was a high prevalence of coexisting asthma and a high level serum IgE level. These characteristics may account for the fragility of tracheal tissues and a predisposition to asthma in patients with mild HPP. The genotypes of the five mild HPP patients in our present study series included 1) compound heterozygous for severe and hypomorphic mutations, and 2) dominant-negative mutations. All of these mutations were at the homodimer interface, but only the dominant-negative mutation was predicted to cause a severe collision effect between the side chains. This may account for varying mechanisms leading to different effects on TNSALP function

Molecular analysis of low‐level mosaicism of the IKBKG mutation using the X Chromosome Inactivation pattern in Incontinentia Pigmenti

Abstract Background Incontinentia pigmenti (IP) is a rare X‐linked disorder affecting the skin and other ectodermal tissues that is caused by mutation of the IKBKG/NEMO gene. Previous studies have reported that the overall mutation detection rate in IP is ~75%. We hypothesized that a low‐level mosaicism existed in the remaining cases. Methods Genomic variations in the IKBKG gene were examined in 30 IP probands and their family members. Standard mutational analyses were performed to detect common deletions, nucleotide alterations, and copy number variations. To assess skewing of the X chromosome inactivation (XCI) pattern, a HUMARA assay was performed. We compared the results of this analysis with phenotype severity. Results Pathogenic variants were identified in 20 probands (66.7%), the rate of detection was suboptimal. The remaining 10 probands tended to manifest a mild phenotype with no skewed X chromosome inactivation that is generally observed in IP patients. Quantitative nested PCR and digital droplet PCR were performed for the 10 patients and mosaicism of the common IKBKG deletion were identified in five patients. Conclusion Overall, we detected 25 IKBKG mutations (83.3%). Determination of the XCI value in advance of mutational analyses for IP could improve the mutation detection rate. Our improved detection rate for these mutations, particularly those with a low‐level mosaicism, may present opportunities for appropriate genetic counseling

G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription

Methylation of DNA and lysine 9 of histone H3 (H3K9) are well-conserved epigenetic marks for transcriptional silencing. Although H3K9 methylation directs DNA methylation in filamentous fungi and plants, this pathway has not been corroborated in mammals. G9a and GLP/Eu-HMTase1 are two-related mammalian lysine methyltransferases and a G9a/GLP heteromeric complex regulates H3K9 methylation of euchromatin. To elucidate the function of G9a/GLP-mediated H3K9 methylation in the regulation of DNA methylation and transcriptional silencing, we characterized ES cells expressing catalytically inactive mutants of G9a and/or GLP. Interestingly, in ES cells expressing a G9a-mutant/GLP complex that does not rescue global H3K9 methylation, G9a/GLP-target genes remain silent. The CpG sites of the promoter regions of these genes were hypermethylated in such mutant ES cells, but hypomethylated in G9a- or GLP-KO ES cells. Treatment with a DNA methyltransferase inhibitor reactivates these G9a/GLP-target genes in ES cells expressing catalytically inactive G9a/GLP proteins, but not the wild-type proteins. This is the first clear evidence that G9a/GLP suppresses transcription by independently inducing both H3K9 and DNA methylation

Epigenetic regulation of mouse sex determination by the histone demethylase Jmjd1a

Developmental gene expression is defined through cross-talk between the function of transcription factors and epigenetic status, including histone modification. Although several transcription factors play crucial roles in mammalian sex determination, how epigenetic regulation contributes to this process remains unknown. We observed male-to-female sex reversal in mice lacking the H3K9 demethylase Jmjd1a and found that Jmjd1a regulates expression of the mammalian Y chromosome sex-determining gene Sry. Jmjd1a directly and positively controls Sry expression by regulating H3K9me2 marks. These studies reveal a pivotal role of histone demethylation in mammalian sex determination