MicroRNAs (miRs) are evolutionarily conserved, non-coding RNA molecules of approximately 21 nucleotides that regulate the expression of genes that are involved in various biological processes, such as cell proliferation and differentiation. Previously, we reported the presence of a heterogeneous population of mRNAs present in the axons and nerve terminals of primary sympathetic neurons to include the nuclear-encoded mitochondrial mRNA coding for COXIV. Sequence analysis of the 3′UTR of this mRNA revealed the presence of a putative binding site for miR-338, a brain-specific microRNA. Transfection of precursor miR-338 into the axons of primary sympathetic neurons decreases COXIV mRNA and protein levels and results in a decrease in mitochondrial activity, as measured by the reduction of ATP levels. Conversely, the transfection of synthetic anti-miR oligonucleotides that inhibit miR-338 increases COXIV levels, and results in a significant increase in oxidative phosphorylation and also norepinephrine uptake in the axons. Our results point to a molecular mechanism by which this microRNA participates in the regulation of axonal respiration and function by modulating the levels of COXIV, a protein which plays a key role in the assembly of the mitochondrial cytochrome c oxidase complex IV.
Background: Missense mutations in the SHANK3 gene have been detected in autism patients. Results: A mutation in the conserved SPN region of Shank3 improves ligand binding to the ankyrin repeats. Conclusion:The SPN domain regulates accessibility of the ankyrin repeats through an intramolecular interaction. Significance: Autism-associated mutations of Shank3 result in gain-of-function with respect to specific interaction partners.
(1) Axons contain numerous mRNAs and a local protein synthetic system that can be regulated independently of the cell body. (2) In this study, cultured primary sympathetic neurons were employed, to assess the effect of local protein synthesis blockade on axon viability and mitochondrial function. (3) Inhibition of local protein synthesis reduced newly synthesized axonal proteins by 65% and resulted in axon retraction after 6 h. Acute inhibition of local protein synthesis also resulted in a significant decrease in the membrane potential of axonal mitochondria. Likewise, blockade of local protein transport into the mitochondria by transfection of the axons with Hsp90 C-terminal domain decreased the mitochondrial membrane potential by 65%. Moreover, inhibition of the local protein synthetic system also reduced the ability of mitochondria to restore axonal levels of ATP after KCl-induced depolarization. (4) Taken together, these results indicate that the local protein synthetic system plays an important role in mitochondrial function and the maintenance of the axon.
Obscurins (∼70 - 870 kDa), encoded by the single OBSCN gene, are cytoskeletal proteins originally identified in striated muscles with structural and regulatory roles. Recently, analysis of 13,023 genes in breast and colorectal cancers identified OBSCN as one of the most frequently mutated genes, implicating it in cancer formation. Herein we studied the expression profile of obscurins in breast, colon, and skin cancer cell lines and their involvement in cell survival. Immunoblot analysis demonstrated significant reduction of obscurin proteins [corrected] in cancer cells, resulting from decreased mRNA levels and/or the presence of mutant transcripts. In normal epithelium, obscurins localize in cytoplasmic puncta, the cell membrane, and the nucleus. Accordingly, subcellular fractionation demonstrated the presence of 2 novel nuclear isoforms of ∼110 and ∼120 kDa. Nontumorigenic MCF10A breast epithelial cells stably transduced with shRNAs targeting giant obscurins exhibited increased viability (∼30%) and reduced apoptosis (∼20%) following exposure to the DNA-damaging agent etoposide. Quantitative RT-PCR further indicated that the antiapoptotic genes BAG4 and HAX1 were up-regulated (1.5- and 1.4-fold, respectively), whereas initiator caspase-9 and death caspase-3 transcripts were down-regulated (0.8- and 0.6-fold, respectively). Our findings are the first to pinpoint critical roles for obscurins in cancer development by contributing to the regulation of cell survival.
The postsynaptic density is the ultrastructural entity containing the neurotransmitter reception apparatus of excitatory synapses in the brain. A recently identified family of multidomain proteins termed Src homology 3 domain and ankyrin repeat-containing (Shank), also known as proline-rich synapse-associated protein/somatostatin receptor-interacting protein, plays a central role in organizing the subsynaptic scaffold by interacting with several synaptic proteins including the glutamate receptors. We used the Nterminal ankyrin repeats of Shank1 and -3 to search for interacting proteins by yeast two-hybrid screening and by affinity chromatography. By cDNA sequencing and mass spectrometry the cytoskeletal protein ␣-fodrin was identified as an interacting molecule. The interaction was verified by pull-down assays and by coimmunoprecipitation experiments from transfected cells and brain extracts. Mapping of the interacting domains of ␣-fodrin revealed that the highly conserved spectrin repeat 21 is sufficient to bind to the ankyrin repeats. Both interacting partners are coexpressed widely in the rat brain and are colocalized in synapses of hippocampal cultures. Our data indicate that the Shank1 and -3 family members provide multiple independent connections between synaptic glutamate receptor complexes and the cytoskeleton.The clustering of neurotransmitter receptors and cell adhesion molecules at specific sites of the cell membrane (i.e. the postsynaptic membrane) is brought about by a dense network of submembranous proteins that becomes visible as an electron-dense thickening at the postsynaptic membrane (postsynaptic density (PSD)). 1 PSD proteins are able to anchor and cluster membrane-spanning receptors, cell adhesion molecules, or both via protein-protein interaction domains (1, 2).The search for proteins that are localized at the PSD revealed a family of multidomain proteins termed proline-rich synapse-associated proteins 1 and 2 or somatostatin receptorinteracting protein. For convenience and in accord with the most frequently used nomenclature, we will refer to these proteins as Src homology 3 domain and ankyrin repeat-containing (Shank) proteins (see Scheme 1). These proteins may act as a molecular interface between neurotransmitter receptors and cell adhesion molecules and the actin-based cytoskeleton (5, 7, 9). To date three members of this family, Shank1-3, have been identified (4 -11), containing multiple protein-protein interaction domains including ankyrin repeats, an Src homology 3 (SH3) domain, a PSD-95/discs large/ZO-1 (PDZ) domain, several proline-rich regions, and a C-terminal sterile ␣-motif domain (Scheme 1). The highly conserved PDZ domain has been shown to interact with the C terminus of several different proteins such as the postsynaptic density protein synapseassociated protein-associated protein/guanylate kinase-associated protein (4, 6, 9) and several G-protein-coupled receptors, including the somatostatin receptor subtype 2 (5, 10) and the calcium-independent receptor for ␣-latrotoxin (1...
OBJECTIVE-Suppressors of cytokine signaling (SOCS) are implicated in the etiology of diabetes, obesity, and metabolic syndrome. Here, we show that some SOCS members are induced, while others are constitutively expressed, in retina and examine whether persistent elevation of SOCS levels in retina by chronic inflammation or cellular stress predisposes to developing insulin resistance in retina, a condition implicated in diabetic retinopathy.RESEARCH DESIGN AND METHODS-SOCS-mediated insulin resistance and neuroprotection in retina were investigated in 1) an experimental uveitis model, 2) SOCS1 transgenic rats, 3) insulin-deficient diabetic rats, 4) retinal cells depleted of SOCS6 or overexpressing SOCS1/SOCS3, and 5) oxidative stress and light-induced retinal degeneration models. RESULTS-Weshow that constitutive expression of SOCS6 protein in retinal neurons may improve glucose metabolism, while elevated SOCS1/SOCS3 expression during uveitis induces insulin resistance in neuroretina. SOCS-mediated insulin resistance, as indicated by its inhibition of basally active phosphoinositide 3-kinase/AKT signaling in retina, is validated in retinaspecific SOCS1 transgenic rats and retinal cells overexpressing SOCS1/SOCS3. We further show that the SOCS3 level is elevated in retina by oxidative stress, metabolic stress of insulin-deficient diabetes, or light-induced retinal damage and protects ganglion cells from apoptosis, suggesting that upregulation of SOCS3 may be a common physiologic response of neuroretinal cells to cellular stress. CONCLUSIONS-Our data suggest two-sided roles of SOCS proteins in retina. Whereas SOCS proteins may improve glucose metabolism, mitigate deleterious effects of inflammation, and promote neuroprotection, persistent SOCS3 expression caused by chronic inflammation or cellular stress can induce insulin resistance and inhibit neurotrophic factors, such as ciliary neurotrophic factor, leukemia inhibitory factor, and insulin, that are essential for retinal cell survival.
Previously, pA134 was identified as one of the mRNAs present in the squid giant axon. Comparative sequence analyses revealed that the pA134 gene product manifested significant similarity to the mammalian lipoprotein receptor adaptor protein also known as ARH (autosomal recessive hypercholesterolemia). ARH mRNA and protein displayed very similar pattern of expression throughout the mouse brain. Significant levels of expression were observed in cells with a predominantly neuronal profile in the cerebellum, brainstem, olfactory bulb, hippocampus, and cortex. A yeast two hybrid screen for ARH protein interactions in mouse brain identified the following binders: amyloid precursor-like protein 1, low density lipoprotein receptor-related protein (LRP) 1, LRP8, and GABA receptor-associated protein-like 1. The interactions of ARH with LRP1 and GABA receptor-associated protein-like 1 were subsequently verified by co-immunoprecipitation of the protein complexes from transfected human embryonic kidney cells. The presence of ARH mRNA in axon of primary sympathetic neurons was established by RT-PCR analyses and confirmed by in situ hybridization. Taken together, our data suggest that ARH is a multifunctional protein whose spectrum of function in the brain goes beyond the traditionally known metabolism of lipoproteins, and that ARH may be locally synthesized in the axon.
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