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Acetylation of C-terminal lysines modulates protein turnover and stability of Connexin-32
Background
The gap junction protein, Connexin32 (Cx32), is expressed in various tissues including liver, exocrine pancreas, gastrointestinal epithelium, and the glia of the central and peripheral nervous system. Gap junction-mediated cell-cell communication and channel-independent processes of Cx32 contribute to the regulation of physiological and cellular activities such as glial differentiation, survival, and proliferation; maintenance of the hepatic epithelium; and axonal myelination. Mutations in Cx32 cause X-linked Charcot–Marie–Tooth disease (CMT1X), an inherited peripheral neuropathy. Several CMT1X causing mutations are found in the cytoplasmic domains of Cx32, a region implicated in the regulation of gap junction assembly, turnover and function. Here we investigate the roles of acetylation and ubiquitination in the C-terminus on Cx32 protein function. Cx32 protein turnover, ubiquitination, and response to deacetylase inhibitors were determined for wild-type and C-terminus lysine mutants using transiently transfected Neuro2A (N2a) cells.
Results
We report here that Cx32 is acetylated in transfected N2a cells and that inhibition of the histone deacetylase, HDAC6, results in an accumulation of Cx32. We identified five lysine acetylation targets in the C-terminus. Mutational analysis demonstrates that these lysines are involved in the regulation of Cx32 ubiquitination and turnover. While these lysines are not required for functional Cx32 mediated cell-cell communication, BrdU incorporation studies demonstrate that their relative acetylation state plays a channel-independent role in Cx32-mediated control of cell proliferation.
Conclusion
Taken together these results highlight the role of post translational modifications and lysines in the C-terminal tail of Cx32 in the fine-tuning of Cx32 protein stability and channel-independent functions
Hereditary spastic paraplegia is a novel phenotype for GJA12/GJC2 mutations
Recessive mutations in GJA12/GJC2, the gene that encodes the gap junction protein connexin47 (Cx47), cause Pelizaeus-Merzbacher-like disease (PMLD), an early onset dysmyelinating disorder of the CNS, characterized by nystagmus, psychomotor delay, progressive spasticity and cerebellar signs. Here we describe three patients from one family with a novel recessively inherited mutation, 99C>G (predicted to cause an Ile>Met amino acid substitution; I33M) that causes a milder phenotype. All three had a late-onset, slowly progressive, complicated spastic paraplegia, with normal or near-normal psychomotor development, preserved walking capability through adulthood, and no nystagmus. MRI and MR spectroscopy imaging were consistent with a hypomyelinating leukoencephalopathy. The mutant protein forms gap junction plaques at cell borders similar to wild-type (WT) Cx47 in transfected cells, but fails to form functional homotypic channels in scrape-loading and dual whole-cell patch clamp assays. I33M forms overlapping gap junction plaques and functional channels with Cx43, however, I33M/Cx43 channels open only when a large voltage difference is applied to paired cells. These channels probably do not function under physiological conditions, suggesting that Cx47/Cx43 channels between astrocytes and oligodendrocytes are disrupted, similar to the loss-of-function endoplasmic reticulum-retained Cx47 mutants that cause PMLD. Thus, GJA12/GJC2 mutations can result in a milder phenotype than previously appreciated, but whether I33M retains a function of Cx47 not directly related to forming functional gap junction channels is not known
A Large-Scale, Consortium-Based Genomewide Association Study of Asthma
BACKGROUND
Susceptibility to asthma is influenced by genes and environment;
implicated genes may indicate pathways for therapeutic intervention.
Genetic risk factors may be useful in identifying subtypes of asthma and
determining whether intermediate phenotypes, such as elevation of the
total serum IgE level, are causally linked to disease.
METHODS
We carried out a genomewide association study by genotyping 10,365
persons with physician-diagnosed asthma and 16,110 unaffected persons,
all of whom were matched for ancestry. We used random-effects pooled
analysis to test for association in the overall study population and in
subgroups of subjects with childhood-onset asthma (defined as asthma
developing before 16 years of age), later-onset asthma, severe asthma,
and occupational asthma.
RESULTS
We observed associations of genomewide significance between asthma and
the following single-nucleotide polymorphisms: rs3771166 on chromosome
2, implicating IL1RL1/IL18R1 (P = 3x10(-9)); rs9273349 on chromosome 6,
implicating HLA-DQ (P = 7x10(-14)); rs1342326 on chromosome 9, flanking
IL33 (P = 9x10(-10)); rs744910 on chromosome 15 in SMAD3 (P = 4x10(-9));
and rs2284033 on chromosome 22 in IL2RB (P = 1.1x10(-8)). Association
with the ORMDL3/GSDMB locus on chromosome 17q21 was specific to
childhood-onset disease (rs2305480, P = 6x10(-23)). Only HLA-DR showed a
significant genomewide association with the total serum IgE
concentration, and loci strongly associated with IgE levels were not
associated with asthma.
CONCLUSIONS
Asthma is genetically heterogeneous. A few common alleles are associated
with disease risk at all ages. Implicated genes suggest a role for
communication of epithelial damage to the adaptive immune system and
activation of airway inflammation. Variants at the ORMDL3/GSDMB locus
are associated only with childhood-onset disease. Elevation of total
serum IgE levels has a minor role in the development of asthma