2007 — 2020 |
Boehning, Darren F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Mechanisms of Apoptotic Calcium Signaling @ University of Texas Hlth Sci Ctr Houston
DESCRIPTION (provided by applicant): Apoptotic calcium release from inositol 1,4,5-trisphosphate receptors (IP3Rs) regulates cell death in response to multiple stimuli, including engagement of the Fas receptor. We have found that the Fas receptor (FasR) directly engages and activates components of the T cell receptor complex (TCR) to elicit apoptotic calcium release from IP3R channels. However, the underlying molecular mechanisms remain to be established. Our preliminary data suggests that Fas and TCR components are recruited into detergent-resistant membrane microdomains enriched in cholesterol and glycosphingolipids (lipid rafts) following Fas stimulation. Furthermore, our data indicates that this is associated wih the rapid stimulus-dependent palmitoylation of the Src family kinase Lck. The central hypothesis of this proposal is that apoptotic calcium release is mediated by rapid palmitoylation of components of the Fas receptor complex, recruitment into lipid rafts, and subsequent activation of TCR components. We will investigate this hypothesis in two Specific Aims. In Specific Aim 1, we will determine if recruitment of the FasR/TCR supramolecular signaling complex into lipid rafts mediates Fas-associated apoptotic calcium release. In Specific Aim 2: Determine if rapid stimulus-dependent palmitoylation/depalmitoylation of Lck by DHHC21 mediates Fas-associated apoptotic calcium release. We will use a variety of molecular biological, biochemical, and novel live cell imaging techniques to determine the mechanisms by which Fas receptor engages the TCR complex to mediate T lymphocyte calcium release and cell death. These studies may uncover novel therapeutic targets for diseases associated with altered T cell homeostasis.
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0.941 |
2008 — 2010 |
Barral, Jose Manuel (co-PI) [⬀] Boehning, Darren F |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Ubiquilin and Alzheimer's Disease @ University of Texas Medical Br Galveston
DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is the most common neurodegenerative disorder associated with aging. Insight into the molecular basis of AD progression has been facilitated by the study of rare familial mutations which result in altered proteolytic processing of amyloid precursor protein (APP). One of the proteolytic products of APP, A2 peptide, is prone to aggregation and likely contributes to neuronal loss and cognitive dysfunction in AD. Unlike familial cases of AD, the underlying causes of late-onset AD are poorly understood. Genetic studies indicate that the UBQLN1 gene may be linked to late-onset AD. The protein product of the UBQLN1 gene, ubiquilin-1, is a modular protein which contains ubiquitin-like and ubiquitin-associated domains. Ubiquilin-1 also contains Sti1 domains implicated in protein-protein interactions and co-chaperone activity. These domains are present in several co-chaperone proteins, some of which have intrinsic molecular chaperone activity. A chaperone function for ubiquilin has not been demonstrated. We have found that ubiquilin has intrinsic molecular chaperone activity and binds to the cytosolic domain of APP. This binding directly modulates both the maturation and degradation of APP. The central hypothesis of this proposal is that ubiquilin is a key quality control molecule for APP folding and maturation: ubiquilin binds to and exerts chaperone activity on folding-competent APP species along the secretory pathway, whereas it targets misfolded APP to the proteasome system for degradation. This hypothesis will be addressed in three Specific Aims. In the first Aim, we will determine whether ubiquilin functions as a molecular chaperone for APP or APP fragments. In the second Aim, we will determine whether ubiquilin targets APP and/or APP proteolytic fragments for proteasomal degradation. Finally, in the third Aim we will determine whether ubiquilin modulates APP maturation and subsequent toxicity. Biochemical assays and live cell imaging will be used to determine how ubiquilin modulates APP maturation and degradation in cell lines and primary cortical neurons. We propose a novel function for ubiquilin as a molecular chaperone and hypothesize this function is critical for regulating the maturation, degradation and toxicity of APP. These studies may provide fundamental insights into the mechanistic basis of ubiquilin function in late-onset Alzheimer's disease. PUBLIC HEALTH RELEVANCE: Alzheimer's disease (AD) is the most prevalent cause of dementia associated with aging. The molecular basis of AD progression in the aging population is poorly understood. This proposal will address the physiologic mechanisms by which the UBQLN1 gene product modulates amyloid precursor protein aggregation and toxicity, thus potentially revealing new therapeutic targets for this common disease.
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0.941 |
2019 — 2021 |
Akimzhanov, Askar Boehning, Darren F |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Regulation of Calcium Signaling by Protein Lipidation
ABSTRACT Intracellular calcium signals play a vital role in regulating immune system homeostasis and function. In T cells, calcium ions serve as a critical second messenger in a broad variety of cellular processes regulating T cell activation, proliferation and differentiation of naïve T cells into effector or memory cells. The mechanism supporting the sustained calcium influx into the cytoplasm is known as store-operated calcium entry (SOCE). Two proteins, Orai1 and STIM1, were identified as primary modulators of SOCE in T cells. SOCE is initiated when STIM1 senses the depletion of internal calcium stores and associates with the pore-forming Orai1 to assemble the calcium release-activated channel. The critical role of STIM1 and Orai1 in the regulation of T cell immune responses is well supported by genetic studies performed in animals as well as clinical data. Biological consequences of Orai1 or STIM1 deficiencies include severe immunodeficiency, tubular aggregate myopathy, and Stormorken syndrome. In our preliminary experiments, we have identified both Orai1 and STIM1 as endogenously S-acylated proteins. S-acylation, a reversible post-translational lipidation of cysteine residues with long-chain fatty acids, is catalyzed by the family of DHHC palmitoyl acyltransferases known to regulate the function of many key T cell signaling proteins. Our previous studies strongly suggest that stimulus-dependent protein lipidation is an essential part of the intricate signaling machinery controlling T cell activation and function. Therefore, we hypothesize that dynamic S-acylation of Orai1 and STIM1 is a critical regulator of calcium entry in T cells. To uncover the role of protein lipidation in calcium signaling, we will (1) determine whether Orai1 and STIM1 are S-acylated proteins in T cells, (2) determine the functional consequences of Orai1 and STIM1 acylation and (3) identify the enzymatic mechanisms mediating Orai1 and STIM1 S-acylation. The successful completion of the proposed project will demonstrate the biological significance of protein lipidation in regulation of SOCE as well as the role of palmitoyl acyltransferases in regulation of the calcium signaling in T cells with relevance to primary immunodeficiency disease.
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0.923 |