26 research outputs found
Integration Of Extracellular And Intracellular Signals Via The Calcium Sensing Receptor (CASR)
The Ca2+-sensing receptor (CaSR) regulates the calcium homeostasis in the human body via sensing fluctuations in the extracellular Ca2+ concentration. Naturally occurring mutations in the CaSR could result in Ca2+ regulation disorders. In the present study, we use several complementary approaches including imaging [Ca2+]i response in living cells at the cellular level and using molecular dynamic (MD) simulations at the atomic level to provide important insights into the behavior of the receptor in both normal and disease statuses. We demonstrated that the molecular connectivity between [Ca2+]oâbinding sites is responsible for the functional positive homotropic cooperativity in the CaSRâs response to [Ca2+]o. Naturallyoccurring disease mutations near Site 1 disrupted the cooperativity. We further identified an L-Phe-binding pocket adjacent to Ca2+-binding Site 1, which is essential for functional positive heterotropic cooperativity by having a global impact on all five of the predicted Ca2+-binding sites in the ECD with regards to [Ca2+]o-evoked [Ca2+]i signaling. Furthermore, the CaSRâs ECDs have been expressed using both bacteria and mammalian systems and were characterized using the fluorescence titration spectroscopy, circular dichroism technique as well as the NMR spectroscopy. Our studies show calcium and Phe directly bind to the ECD domain directly and interactively. Moreover, we also demonstrated that intracellular trafficking of the CaSR is a complex process, which involves modulation by calmodulin and can possibly be affected by different CaSR isoforms when expressing in various cell lines. The studies on the isolated proteins will pave the way for future protein crystallization and related structural research
The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator
BACKGROUND: Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. RESULTS: Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of ÎF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant ÎF508 CFTR. CONCLUSION: These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR
Phagocytosis and signaling in the innate immune system
The innate immune response provides broad spectrum defense through germline encoded components. Many aspects of innate immunity, such as the activation of NFκB transcription factors and phagocytosis, are highly conserved within the animal kingdom. The innate immune response of the cow, in particular, is important due to the cow's agricultural value. A major proportion of acute disease in domestic cattle is caused by Gram-negative bacteria, which produce the outer membrane component lipopolysaccharide (LPS). LPS binds to Toll-like receptor (TLR) 4 and activates multiple signaling pathways, which have been well-studied in humans, but not in ruminants. Human myeloid differentiation-factor 88 (MyD88) and TIR-domain containing adaptor protein (TIRAP) are critical proteins in the LPS-induced NFκB and apoptotic signaling pathways in humans. We demonstrated through the expression of dominant negative constructs in bovine endothelial cells that both MyD88 and TIRAP activate NFκB in the cow. Additionally, bovine TIRAP was also shown to transduce LPS–induced apoptosis, indicating that multiple aspects of the TLR4–dependent signaling pathways are conserved between cows and humans. The model organism Drosophila melanogaster, was subsequently utilized to investigate the role of another branch of the innate immune response: phagocytosis. The extracellular fluid surrounding phagocytic cells in Drosophila has a high concentration of the amino acid glutamate. While glutamate has been well-characterized as a neurotransmitter, its effect, if any, on immune cells is largely unknown. We identified that a putative glutamate transporter in D. melanogaster, polyphemus (polyph), is critical to the fly's immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles but not of latex beads. Additionally, polyph flies show increased susceptibility to S.aureus infection, decreased induction of the antimicrobial peptide (AMP) Cecropin, increased melanization response, and increased ROS production. Glutamate transport has previously been shown to regulate the synthesis of the antioxidant glutathione. We demonstrate that a polyph–dependent redox system is necessary to maintain the immune cells' function against an infection. By utilizing two species, the cow and the fly, to study the innate immune system, we have gained unique and novel insights into NFκB activation and phagocytosis
Learning and adaptation in the Drosophila olfactory system
All forms of memory rely on plasticity to correctly store information. Plasticity ensures that certain sets of cells consistently activate each other through the strengthening of certain synapses while others are weakened. This plasticity ensures that a stimulus (internally or externally generated) consistently activates the same cells to produce the same response. These plastic changes can be short-lived, lasting mere seconds, or longer-lived, lasting days, weeks, and months. To ensure accurate storage of memory tight regulation is required. Mammalian memory systems are complex, involving the integration and processing of information from a wide variety of areas. To overcome the issue of complexity memory is often studied in less complex organisms. One of the leading models for memory is the Drosophila olfactory system. In Drosophila, associative memories are formed in the Mushroom Body (MB). Over the past 30 years, work in the MB has given us massive insight into plasticity processes and the tight regulation required to ensure accurate coding. Here I aim to test the limits of this regulation and identify novel mechanisms that may ensure accurate memory storage.
First, I tested how robust activity regulation in the mushroom body is and how well it adapts to a challenge that it could face in nature,i.e, overactivity induced by pesticides in a food source. Using pesticides that increase cholinergic signaling, I hoped to identify the maximum dose a fly can ingest and still accurately encode associative memory. Furthermore, I wanted to see if the system could adapt to this disruption over time to restore functionality. In the end, I could not find a dose that significantly disrupted learning before fly death, showing that olfactory regulation is at least as robust than other vital systems.
Second, one of the major players in memory storage in mammals is nitric oxide (NO). It has also been identified as a factor in the pathogenesis of many neurological conditions. Until recently, no such role in memory had been identified in flies. I wanted to see if manipulating NO levels could disrupt memory and, if so, what effect NO had at a cellular level. Though behavioral experiments were inconclusive, I show here that mushroom body survival was reduced after high doses of the nitric oxide donor s-nitrosoglutathione (GSNO).
Finally, I looked at a more everyday challenge to the olfactory system, complex odor mixtures. I used the αâČ3 mushroom body output neuron (MBON), which signals novelty, to show that suppression of certain cells of the mushroom body (MB) makes the components of a mixture appear novel compared to a mixture. This suppression could provide a mechanism by which flies can learn which part of a mixture is predictive of the reward or punishment. Furthermore, I show evidence that different Kenyon cell (KC) subtypes may code mixtures differently depending on the complexity of the odor environment
Investigating the role of optineurin in bone biology and Paget's disease of bone
Pagetâs disease of bone (PDB) is a common disease with a strong genetic
component. Approaches such as linkage analysis and candidate gene studies have
shown that mutations in Sequestosome 1 (SQSTM1) explain up to 40% of familial
cases and 10% of sporadic cases, however the majority of PDB patients have no
mutations in this gene. Genome-wide association studies (GWAS) have recently
identified new susceptibility loci for PDB including variants at CSF1, TNFRSF11A,
OPTN, TM7SF4, PML, NUP205 and RIN3 loci. These loci were confirmed to be
associated with PDB in various European populations. OPTN encodes optineurin, a
widely expressed protein involved in many cellular processes but its role in bone
metabolism is yet unknown. The aim of this PhD thesis was to investigate the role of
OPTN in bone metabolism and PDB using in vitro and in vivo studies. In chapter 3,
the OPTN rs1561570 identified by previous GWAS was examined for its association
with the severity and clinical outcome of PDB in patients without SQSTM1 mutations.
The results showed that rs1561570 was significantly associated with total disease
severity score so that carriers of the risk allele âTâ had higher severity score compared
to non-carriers (P < 0.05). A trend for reduced quality of life physical scores (SF36)
was also associated with the rs1561570 risk allele, but the relationship was not
statistically significant. In order to identify functional variants within OPTN, the
coding regions as well as the exon-intron boundaries were sequenced in 24 familial
PDB cases and 19 controls. No mutation was found that could be predicted as
pathogenic suggesting that disease susceptibility could be mediated by regulatory
polymorphisms that influence gene expression. In chapter 4, the role of OPTN was
investigated in osteoclast development using in vitro knockdown experiments. Optn
was expressed in mouse bone marrow derived macrophages (BMDMs) as well as all
stages of osteoclast development and it was significantly increased three days post
RANKL treatment. Optn expression was knocked down in BMDMs and cells were
induced to form osteoclast in the presence of RANKL and M-CSF. Compared to non-targeted
cells, Optn depleted cells formed significantly more and larger osteoclasts (P<
0.05). Optn knockdown was also found to enhance osteoclast survival as well as
RANKL-induced NFÎșB activation. In chapter 5, the role of OPTN was investigated in
vitro from cells obtained from knock in mice with a loss-of-function mutation in Optn
(OptnD477N/D477N). In agreement with the in vitro knockdown experiments, osteoclasts
were significantly higher and larger in mutant mice compared to WT and the NF-B
activity measured by luciferase reporter assay was significantly higher in cells from
OptnD477N/D477N compared to WT during most stages of osteoclast development. OPTN
from mutant and WT mice was co-precipitated with its CYLD binding-partner, which
acts as a negative regulator to RANK signalling by inhibiting the TRAF6 downstream
signalling. The data from this immunoprecipitation (IP) experiment revealed that
defective OPTN interacted less with CYLD from mutant mice compared to WT. This
study also showed that OPTN was expressed in osteoblasts and the expression rate did
not change during osteoblast development. The data obtained from the mineralization
assay revealed no significant difference between OptnD477N/D477N and WT. In chapter
6, I investigated the effect of the D477N loss of function mutation in Optn on bone
metabolism. Bone Histomorphometrical analysis of OptnD477N/D477N mice showed
higher bone resorption parameters (Oc.N/BS and Oc.S/BS) compared to wild type
(WT). Osteoid analysis showed evidence of increased bone formation parameters
(OS/BS and OV/BV) in mutant mice compared to WT. Calcein labelling showed a
significant difference in mineral apposition rate (MAR) from mutant mice compared
to WT. Analysis of serum biomarkers of bone turnover showed evidence of enhanced
bone turnover in mutant mice compared to WT. Micro computed tomography (ÎŒCT)
analysis of 4 and 14 months old mice showed no significant differences in bone
morphology between WT and OptnD477N/D477N mice of both sexes.
In conclusion, this study has shown for the first time that OPTN plays a role in
regulating bone turnover by acting as a negative regulator of osteoclast differentiation.
The data obtained from this study strongly suggest the crucial role of OPTN in RANK
signalling. The effect of OPTN on osteoblast activity may be direct or indirect
compensation for increased osteoclast activity. Further detailed studies will be
required to explore the underlying mechanism of OPTN including downstream RANK
signalling and a complete knockout model to corroborate these findings
Mitochondrial calcium uptake and release mechanisms as key regulators of cell life or death
Mitochondria are cellular organelles that play a key role in several physiological processes, including cell proliferation, differentiation, cell death and the regulation of cellular calcium (Ca2+) homeostasis.
Increases in mitochondrial Ca2+ activate several dehydrogenases and carriers, inducing enhance in the respiratory rate, H+ extrusion, and ATP production necessary for the correct energy state of the cell.
The mitochondrial Ca2+ uptake and release mechanisms are based on the utilization of gated channels for Ca2+ uptake and exchangers for release that are dependent upon the negative mitochondrial membrane potential, which represents the driving force for Ca2+ accumulation in the mitochondrial matrix.
In this thesis, the attention was focused on two mechanisms in particular, the mitochondrial Ca2+ influx system by the activity of Mitochondrial Calcium Uniporter (MCU) complex, and the high-conductance channel mitochondrial Permeability Transition Pore (mPTP), responsible for a state of non-selective permeability of the inner mitochondrial membrane (IMM); its opening in non-physiological conditions leads to Ca2+ release from mitochondria and triggers cell death mechanisms. Thus the maintenance of the mitochondrial Ca2+ homeostasis is essential for a proper balance between cell life or death.
In particular it will be discussed the possible involvement of MCU in the cell cycle, as the Ca2+ accumulation by MCU is important for the regulation of cell life and energy production. It will be shown that MCU is mainly expressed in specific phases of the cell cycle and this expression positive correlates with the mitochondrial membrane potential. MCU overexpression instead does not alter cell cycle phases.
It will also described the role of the c subunit of Fo ATP synthase in mitochondrial permeability transition (MPT) and it will be demonstrated to be a critical component of the mPTP complex. Finally it will be discussed the role of mPTP in mitochondrial Ca2+ efflux and it will be shown that it is a dispensable element for mitochondrial Ca2+ efflux in non-pathological conditions
RÎle de la protéine Arc (Activity-regulated cytoskeleton-associated protein) dans les adaptations moléculaires et comportementales induites par la cocaïne
Molecular and cellular adaptations induced by drugs of abuse in the reward system play a key role in long-term behavioral alterations encountered in addiction. This work falls within an approach of understanding the cellular processes rapidly engaged by cocaine that could underlie the persistent alteration of neuronal physiology and behaviors. Arc protein is a major player in neuronal plasticity. Arc is induced in many behavioral paradigms and is essential for long-term synaptic plasticity and memory consolidation. The aim of this study was to characterize the profile and modality of Arc induction within the mouse striatum in response to cocaine administration. Our study shows that Arc expression is rapidly and transiently increased in the striatum after acute cocaine in an ERK-dependent fashion. This work revealed that cocaine-induced Arc protein rapidly and transiently accumulates in the nucleus of striatal neurons. In the nucleus, Arc is preferentially expressed in active transcription regions and localizes at the vicinity of phosphorylated histones H3. In vitro Arc overexpression decreased glutamate-induced Histones H3 phosphorylation showing that Arc interferes with activity-dependent chromatin remodeling. In vivo genetic invalidation of Arc expression in a transgenic mouse model was associated with a decreased chromatin compaction and increased RNA Polymerase II activity suggesting a repressive role of Arc on transcriptional mechanisms. Total Arc loss of expression leads to increased sensitivity to cocaine and promotes long-term behavioral alterations induced by low doses of cocaine.Les adaptations cellulaires et molĂ©culaires induites par les drogues jouent un rĂŽle central dans les altĂ©rations comportementales Ă long terme observĂ©es dans lâaddiction. Cette Ă©tude sâinscrit dans une dĂ©marche de comprĂ©hension des processus cellulaires rapidement mis en jeu par la cocaĂŻne et susceptibles dâimpacter durablement le fonctionnement neuronal et les comportements. La protĂ©ine Arc joue un rĂŽle clĂ© dans lâĂ©tablissement de la plasticitĂ© synaptique Ă long-terme et la consolidation de la mĂ©moire. Cette Ă©tude visait Ă caractĂ©riser lâinduction de Arc dans le striatum en rĂ©ponse Ă la cocaĂŻne et dâanalyser son rĂŽle dans les rĂ©ponses molĂ©culaires et comportementales quâelle induit. Notre Ă©tude a montrĂ© que lâexpression de Arc est augmentĂ©e rapidement et transitoirement dans le striatum aprĂšs une injection de cocaĂŻne sous la dĂ©pendance de lâactivation de la voie ERK. Nous montrons que la cocaĂŻne induit une forte accumulation de la protĂ©ine Arc dans le noyau des neurones striataux oĂč Arc se localise dans des zones actives de transcription, Ă proximitĂ© des histones H3 phosphorylĂ©es. In vitro, la surexpression de Arc diminue la phosphorylation des histones H3 induite par le glutamate indiquant quâelle altĂšre le remodelage de la chromatine. Lâinvalidation gĂ©nĂ©tique de la protĂ©ine in vivo dans un modĂšle de souris transgĂ©nique conduit Ă une dĂ©compaction de la chromatine associĂ©e Ă une augmentation de lâactivitĂ© de la RNA Polymerase II dĂ©montrant que Arc exerce un effet rĂ©presseur sur les mĂ©canismes transcriptionnels. La perte totale dâexpression de Arc favorise le dĂ©veloppement dâaltĂ©rations comportementales Ă long terme chez des animaux exposĂ©s Ă de faibles doses de cocaĂŻne
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Structure and Function of Glutamate Receptor-Like Channels (GLRs)
Glutamate is essential for proper brain function as it is our nervous systems principal excitatory neurotransmitter, a signal that stimulates nerve cells to send messages to other cells.
Glutamate activates ionotropic glutamate receptors (iGluRs), which are linked to several neurological diseases in cases when they are improperly regulated. iGluRs are transmembrane channels that allow calcium, as well as other cations, into the post synaptic neuron upon binding of glutamate or other agonists.Interestingly, iGluR homologs in plants also mediate calcium signaling upon glutamate activation and were accordingly named glutamate receptor-like channels (GLRs). Cell signaling is critical for plant survival to mediate rapid response to growth, defense, and other environmental cues.
GLRs are found in all plants and vital for their health, hardiness, and adaptation for growth and survival in unfavorable conditions, such as drought, nutrient poor soil, temperature extremes, pathogens, and predators. Plant research is important with vast applications. Firstly, crops are our primary source of nutrition. In addition, plants are used as sources of drugs that we employ for treating diseases. Some examples of plant-derived neuroactive compounds include caffeine in coffee beans, nicotine in tobacco, and opium from poppy plants. In short, optimizing plant growth is beneficial to maintaining our own survival and potentially achievable by understanding GLRs role in plant health and hardiness. Despite their importance for cell signaling and implication in plant defense and regeneration, the structural basis underlying the function of these channels remains ambiguous, representing a critical barrier to our understanding of GLR function.
To address this problem, I dedicated my thesis work to study the structure of GLRs and gain insight into their function. There are 20 GLRs in the model plant organism, Arabidopsis thaliana, classified into 3 different clades (AtGLR1-3). To narrow down which AtGLRs to focus our structural studies on, we investigated clade 3 representatives, as many of these GLR3s have been extensively studied in different plant species, especially crops. For example, studying AtGLR3.4 could provide useful information to how the homolog in rice, OsGLR3.4, contributes to growth and production in rice. Studying AtGLR3.4âs structure may elucidate how agonistic or antagonistic targets bind and gate the channel, potentially revealing âdruggableâ targets to alter plant response for defense and regeneration.
Without any structural information available for GLRs, I started my studies by first focusing on their mammalian homologs, iGluRs. I first designed multiple constructs for heterologous expression and purification from cell culture (for example HEK293S GnTI- cells). Then, I optimized protein extraction and purification to obtain pure protein samples. Purified proteins were then subjected to cryo-electron microscopy (cryo-EM) which eventually allowed us to solve the structure of AtGLR3.4, the first full-length GLR structure.
AtGLR3.4âs structure revealed similarities to structures of its mammalian homologs, iGluRs. In comparison to iGluRs, our GLR structure also showed tetrameric subunit assembly, with a three-layer architecture that includes the ligand binding domain (LBD) in the middle, sandwiched between the extracellular amino terminal domain (ATD) at the top and the transmembrane domain (TMD) at the bottom. In contrast to the majority of iGluR structures, however, AtGLR3.4 displayed unique symmetry and domain arrangement with the non-swapped extracellular ATD and LBD domains. We also provided further evidence supporting ligand binding promiscuity that was previously revealed in isolated LBD crystal structures from other AtGLR3s. Surprisingly, we found endogenous glutathione bound to the ATDs and demonstrated its contribution to channel activity.
It is important to fill the gaps in knowledge about GLR structure to understand how these channels are activated and gated. In doing so, we will learn more about iGluRs as well as better understand plant defense and growth, which has the potential to enhance crop production for food security and our overall survival
Protein Kinases
Proteins are the work horses of the cell. As regulators of protein function, protein kinases are involved in the control of cellular functions via intricate signalling pathways, allowing for fine tuning of physiological functions. This book is a collaborative effort, with contribution from experts in their respective fields, reflecting the spirit of collaboration - across disciplines and borders - that exists in modern science. Here, we review the existing literature and, on occasions, provide novel data on the function of protein kinases in various systems. We also discuss the implications of these findings in the context of disease, treatment, and drug development