Binding EF-hand Domains Research Articles

Recognition of Aedes aegypti Mosquito Saliva Protein LTRIN by the Human Receptor LTβR for Controlling the Immune Response.

Salivary proteins from mosquitoes have received significant attention lately due to their potential to develop therapeutic treatments or vaccines for mosquito-borne diseases. Here, we report the characterization of LTRIN (lymphotoxin beta receptor inhibitor), a salivary protein known to enhance the pathogenicity of ZIKV by interrupting the LTβR-initiated NF-κB signaling pathway and, therefore, diminish the immune responses. We demonstrated that the truncated C-terminal LTRIN (ΔLTRIN) is a dimeric protein with a stable alpha helix-dominant secondary structure, which possibly aids in withstanding the temperature fluctuations during blood-feeding events. ΔLTRIN possesses two Ca2+ binding EF-hand domains, with the second EF-hand motif playing a more significant role in interacting with LTβR. Additionally, we mapped the primary binding regions of ΔLTRIN on LTβR using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and identified that 91QEKAHIAEHMDVPIDTSKMSEQELQFHY118 from the N-terminal of ΔLTRIN is the major interacting region. Together, our studies provide insight into the recognition of LTRIN by LTβR. This finding may aid in a future therapeutic and transmission-blocking vaccine development against ZIKV.

Interaction between the mitochondrial adaptor MIRO and the motor adaptor TRAK

MIRO (mitochondrial Rho GTPase) consists of two GTPase domains flanking two Ca2+-binding EF-hand domains. A C-terminal transmembrane helix anchors MIRO to the outer mitochondrial membrane, where it functions as a general adaptor for the recruitment of cytoskeletal proteins that control mitochondrial dynamics. One protein recruited by MIRO is TRAK (trafficking kinesin-binding protein), which in turn recruits the microtubule-based motors kinesin-1 and dynein-dynactin. The mechanism by which MIRO interacts with TRAK on the mitochondrial membrane is not well understood. Here, we map and quantitatively characterize the interaction of human MIRO1 and TRAK1 and test its potential regulation by Ca2+ and/or GTP binding to MIRO1. TRAK1 binds MIRO1 with low micromolar affinity. The interaction was mapped to a fragment comprising MIRO1’s EF-hands and C-terminal GTPase domain and to a conserved sequence motif within TRAK1 residues 394-431, immediately C-terminal to the Spindly motif. This sequence is sufficient for MIRO1 binding in vitro and is necessary for MIRO1-dependent localization of TRAK1 to mitochondria in cells. MIRO1’s EF-hands bind Ca2+ with dissociation constants (KD) of 3.9 μM and 300 nM. This suggests that under cellular conditions one EF-hand may be constitutively bound to Ca2+ whereas the other EF-hand binds Ca2+ in a regulated manner, depending on its local concentration. Yet, the MIRO1-TRAK1 interaction is independent of Ca2+ binding to the EF-hands and of the nucleotide state (GDP or GTP) of the C-terminal GTPase. The interaction is also independent of TRAK1 dimerization, such that a TRAK1 dimer can be expected to bind two MIRO1 molecules on the mitochondrial surface.

High Expression of Nucleobindin-2 Is Associated With Poor Prognosis in Gastric Cancer

Nucleobindin-2 (NUCB2) is a 396-amino acid protein, cleaved into the N-terminal nesfatin-11-82, nesfatin-285-163 and the C-terminal nesfatin-3166-396. NUCB2 contains a signal peptide, a leucine zipper structure, two Ca2+ binding EF-hand domains, and has a wide variety of basic cellular functions. NUCB2 is also a precursor protein of nesfatin-1, which was originally identified in hypothalamic nuclei, and which is a regulatory factor involved in the central control of food intake and energy balance. There are several reports indicating that NUCB2 is also expressed in various human peripheral tissues. Moreover, recent studies have reported that high levels of NUCB2 mRNA and protein are a potent prognostic factor for prostate cancer, endometrial carcinoma, and breast cancer. NUCB2 was also identified as a potential tumor antigen eliciting autoantibody responses in 5.4% of gastric cancer patients but not in the healthy individuals. However, theclinicopathological significance of NUCB2 expression in gastric cancer has still not been elucidated. Therefore, we examined NUCB2 expression in a large number of gastric cancer patients, using immunohistochemistry, to explore its clinicopathological significance. To explore this, we aimed to investigate the NUCB2 expression in gastric cancer tissues and adjacent non-tumor tissues and its potential relevance to clinicopathological factors and prognosis using immunohistochemistry analysis. In our study, NUCB2 level in gastric cancer tissues was higher than in non-tumor tissues. A high expression of NUCB2 is significantly associated with tumor depth, lymph node metastasis, lymphatic invasion, venous invasion and clinical stage. Furthermore, the expression level of NUCB2 protein was independent predictor of progression-free survival. In summary, NUCB2 might play a crucial role in gastric cancer development and could serve as an independent predictor of prognosis of gastric cancer patients.

Nucleobindin-2 consists of two structural components: The Zn2+-sensitive N-terminal half, consisting of nesfatin-1 and -2, and the Ca2+-sensitive C-terminal half, consisting of nesfatin-3

Nucleobindin-2 (Nucb2) is a protein that has been suggested to play roles in a variety of biological processes. Nucb2 contains two Ca2+/Mg2+-binding EF-hand domains separated by an acidic amino acid residue-rich region and a leucine zipper. All of these domains are located within the C-terminal half of the protein. At the N-terminal half, Nucb2 also possesses a putative Zn2+-binding motif. In our recent studies, we observed that Nucb2 underwent Ca2+-dependent compaction and formed a mosaic-like structure consisting of intertwined disordered and ordered regions at its C-terminal half. The aim of this study was to investigate the impact of two other potential ligands: Mg2+, which possesses chemical properties similar to those of Ca2+, and Zn2+, for which a putative binding motif was identified. In this study, we demonstrated that the binding of Mg2+ led to oligomerization state changes with no significant secondary or tertiary structural alterations of Nucb2. In contrast, Zn2+ binding had a more pronounced effect on the structure of Nucb2, leading to the local destabilization of its N-terminal half while also inducing changes within its C-terminal half. These structural rearrangements resulted in the oligomerization and/or aggregation of Nucb2 molecules. Taken together, the results of our previous and current research help to elucidate the structure of the Nucb2, which can be divided into two parts: the Zn2+-sensitive N-terminal half (consisting of nesfatin-1 and -2) and the Ca2+-sensitive C-terminal half (consisting of nesfatin-3). These results may also help to open a new discussion regarding the diverse roles that metal cations play in regulating the structure of Nucb2 and the various physiological functions of this protein.

Abstract 270: Micu1 Regulates Mitochondrial Structural Integrity and Cristae Function Independent of Mcu Calcium Uptake

MICU1 is a Ca 2+ -sensor regulating the mitochondrial Ca 2+ uniporter channel (mtCU) and mitochondrial Ca 2+ uptake. MICU1 contains two Ca 2+ - binding EF-hand domains and directly interacts with the mtCU pore-forming subunit, MCU. MICU1-null mouse and fly models display perinatal lethality with disorganized mitochondrial architecture. Interestingly, these phenotypes are distinct from other mtCU loss-of-function models ( MCU, MICU2, EMRE, MCUR1 ) and thus are likely not explained solely by changes in matrix Ca 2+ content, suggesting MICU1 has cellular functions independent of the mtCU. To discern novel MICU1 molecular interactions we utilized a proximity-based biotinylation proteomic approach, BioID2, and expressed a MICU1-BioID2 fusion construct in wild-type and MCU -/- cells. LC-MS analysis of purified biotinylated proteins identified Mitochondrial Contact Site and Cristae Organizing System (MICOS) components MIC60, CHCHD2, and CHCHD3 as MICU1 interacting partners. Loss of MCU did not affect the MICU1-MICOS interaction, suggesting that MICU1 could be regulating the MICOS independent of the mtCU. Fast protein liquid chromatography (FPLC), blue native-PAGE, co-immunoprecipitation, live-cell Ca 2+ imaging, electron microscopy, confocal and super-resolution imaging confirmed that MICU1 (independent of matrix Ca 2+ ) is regulating mitochondrial membrane dynamics, membrane potential, and cell death signaling. Further, the deletion of Chchd2, a core component of the MICOS, resulted in disruption of cristae structure without any observable effect on mitochondrial Ca 2+ uptake. In addition, RNA sequencing of murine models of heart failure revealed a correlation between the expression of MICU1 and MICOS components and disease progression. These results are the first to identify how Ca 2+ regulates cristae structure and function and suggests MICU1, at the MICOS, regulates mitochondrial membrane remodeling in the context of cardiac stress and disease independent of the mtCU.

MON-313 High Expression of Nucleobindin-2 Is Associated with Poor Prognosis in Gastric Cancer

Nucleobindin-2 (NUCB2) is a 396-amino acid protein, cleaved into the N-terminal nesfatin-11-82, nesfatin-285-163and the C-terminal nesfatin-3166-396. NUCB2 contains a signal peptide, a leucine zipper structure, two Ca2+binding EF-hand domains, and has a wide variety of basic cellular functions. NUCB2 is also a precursor protein of nesfatin-1, which was originally identified in hypothalamic nuclei, and which is a regulatory factor involved in the central control of food intake and energy balance. There are several reports indicating that NUCB2 is also expressed in various human peripheral tissues. Moreover, recent studies have reported that high levels of NUCB2 mRNA and protein are a potent prognostic factor for prostate cancer, endometrial carcinoma, and breast cancer. Suzuki et al.described high NUCB2 immunoreactivity in patients with breast cancer, and NUCB2 protein expression associated significantly with lymph node metastasis and an increased risk of recurrence. They found that NUCB2 significantly increased tumor cell proliferation, migration, and invasion, suggesting that NUCB2 plays a crucial role in the metastatic process of breast cancer. Several researchers have reported that the expression level of NUCB2 was significantly higher in prostate cancer tissues than in non-cancerous tissues, and was associated with lymph node metastasis and shorter survival. However,theclinicopathologicalsignificance of NUCB2 expression in gastric cancer has still not beenelucidated. Therefore, we examined NUCB2 expression in a large number of gastric cancer patients, using immunohistochemistry, to explore its clinicopathologicalsignificance. To explore this, we aimed to investigate the NUCB2 expression in gastric cancer tissues and adjacent non-tumor tissues and its potential relevance to clinicopathological factors and prognosis using immunohistochemistry analysis. In our study, NUCB2 level in gastric cancertissues was higher than in non-tumor tissues. A high expression of NUCB2 is significantly associated with tumor depth, lymph node metastasis, lymphatic invasion, venous invasion and clinical stage. Furthermore, the expression level of NUCB2 protein was independent predictor of progression-free survival. In summary, NUCB2 might play a crucial role in gastric cancerdevelopment and could serve as an independent predictor of prognosis of gastric cancerpatients.

IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells are expanded by dendritic cells silenced for Allograft Inflammatory Factor-1.

Allograft Inflammatory Factor-1 (AIF1) is a cytoplasmic scaffold protein that contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes in immune cells. The protein plays a dominant role in both macrophage- and dendritic cell (DC)-mediated inflammatory responses. This study now reports that AIF1 expression in DC is important in directing CD8+ T cell effector responses. Silencing AIF1 expression in murine CD11c+ DC suppressed antigen-specific CD8+ T cell activation, marked by reduced CXCR3, IFNγ and Granzyme B expression, and restrained proliferation. These primed CD8+ T cells had impaired cytotoxic killing of target cells in vitro. In turn, studies identified that AIF1 silencing in DC robustly expanded IL-10 producing CD8+ CD122+ PD-1+ regulatory T cells that suppressed neighboring immune effector responses through both IL-10 and PD-1-dependent mechanisms. In vivo studies recapitulated bystander suppression of antigen-responsive CD4+ T cells by the CD8+ Tregs expanded from the AIF1 silenced DC. These studies further demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and present a novel target for engineering tolerogenic DC-based immunotherapies.

Calcineurin Regulatory Subunit Calcium-Binding Domains Differentially Contribute to Calcineurin Signaling in Saccharomyces cerevisiae.

The protein phosphatase calcineurin is central to Ca2+ signaling pathways from yeast to humans. Full activation of calcineurin requires Ca2+ binding to the regulatory subunit CNB, comprised of four Ca2+-binding EF hand domains, and recruitment of Ca2+-calmodulin. Here we report the consequences of disrupting Ca2+ binding to individual Cnb1 EF hand domains on calcineurin function in Saccharomyces cerevisiae Calcineurin activity was monitored via quantitation of the calcineurin-dependent reporter gene, CDRE-lacZ, and calcineurin-dependent growth under conditions of environmental stress. Mutation of EF2 dramatically reduced CDRE-lacZ expression and failed to support calcineurin-dependent growth. In contrast, Ca2+ binding to EF4 was largely dispensable for calcineurin function. Mutation of EF1 and EF3 exerted intermediate phenotypes. Reduced activity of EF1, EF2, or EF3 mutant calcineurin was also observed in yeast lacking functional calmodulin and could not be rescued by expression of a truncated catalytic subunit lacking the C-terminal autoinhibitory domain either alone or in conjunction with the calmodulin binding and autoinhibitory segment domains. Ca2+ binding to EF1, EF2, and EF3 in response to intracellular Ca2+ signals therefore has functions in phosphatase activation beyond calmodulin recruitment and displacement of known autoinhibitory domains. Disruption of Ca2+ binding to EF1, EF2, or EF3 reduced Ca2+ responsiveness of calcineurin, but increased the sensitivity of calcineurin to immunophilin-immunosuppressant inhibition. Mutation of EF2 also increased the susceptibility of calcineurin to hydrogen peroxide inactivation. Our observations indicate that distinct Cnb1 EF hand domains differentially affect calcineurin function in vivo, and that EF4 is not essential despite conservation across taxa.

Inhibition of Allograft Inflammatory Factor-1 in Dendritic Cells Restrains CD4+ T Cell Effector Responses and Induces CD25+Foxp3+ T Regulatory Subsets.

Allograft inflammatory factor-1 (AIF1) is a cytoplasmic scaffold protein shown to influence immune responses in macrophages and microglial cells. The protein contains Ca2+ binding EF-hand and PDZ interaction domains important for mediating intracellular signaling complexes. This study now reports that AIF1 is expressed in CD11c+ dendritic cells (DC) and silencing of expression restrains induction of antigen-specific CD4+ T cell effector responses. AIF1 knockdown in murine DC resulted in impaired T cell proliferation and skewed polarization away from T helper type 1 and 17 fates. In turn, there was a parallel expansion of IL-10-producing and CD25+Foxp3+ T regulatory subsets. These studies are the first to demonstrate that AIF1 expression in DC serves as a potent governor of cognate T cell responses and presents a novel target for engineering tolerogenic DC-based immunotherapies.

Annotation of nerve cord transcriptome in earthworm Eisenia fetida

In annelid worms, the nerve cord serves as a crucial organ to control the sensory and behavioral physiology. The inadequate genome resource of earthworms has prioritized the comprehensive analysis of their transcriptome dataset to monitor the genes express in the nerve cord and predict their role in the neurotransmission and sensory perception of the species. The present study focuses on identifying the potential transcripts and predicting their functional features by annotating the transcriptome dataset of nerve cord tissues prepared by Gong et al., 2010 from the earthworm Eisenia fetida. Totally 9762 transcripts were successfully annotated against the NCBI nr database using the BLASTX algorithm and among them 7680 transcripts were assigned to a total of 44,354 GO terms. The conserve domain analysis indicated the over representation of P-loop NTPase domain and calcium binding EF-hand domain. The COG functional annotation classified 5860 transcript sequences into 25 functional categories. Further, 4502 contig sequences were found to map with 124 KEGG pathways. The annotated contig dataset exhibited 22 crucial neuropeptides having considerable matches to the marine annelid Platynereis dumerilii, suggesting their possible role in neurotransmission and neuromodulation. In addition, 108 human stem cell marker homologs were identified including the crucial epigenetic regulators, transcriptional repressors and cell cycle regulators, which may contribute to the neuronal and segmental regeneration. The complete functional annotation of this nerve cord transcriptome can be further utilized to interpret genetic and molecular mechanisms associated with neuronal development, nervous system regeneration and nerve cord function.

Identification of critical amino acid residues and functional conservation of the Neurospora crassa and Rattus norvegicus orthologues of neuronal calcium sensor-1.

Neuronal calcium sensor-1 (NCS-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (Ca2+) binding EF-hand domains. We performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the N-terminal myristoylation, a conserved glutamic acid residue responsible for Ca2+ binding in the third EF-hand (EF3), and an unusual non-conserved amino acid arginine at position 175 in the Neurospora crassa NCS-1. The N. crassa strains possessing the ncs-1 mutant allele of these three amino acid residues showed impairment in functions ranging from growth, Ca2+ stress tolerance, and ultraviolet survival. In addition, heterologous expression of the NCS-1 from Rattus norvegicus in N. crassa confirmed its interspecies functional conservation. Moreover, functions of glutamic acid at position 120, the first Ca2+ binding residue among all the EF-hands of the R. norvegicus NCS-1 was found conserved. Thus, we identified three critical amino acid residues of N. crassa NCS-1, and demonstrated its functional conservation across species using the orthologue from R. norvegicus.

Genome-Wide Identification of Calcium Dependent Protein Kinase Gene Family in Plant Lineage Shows Presence of Novel D-x-D and D-E-L Motifs in EF-Hand Domain.

Calcium ions are considered ubiquitous second messengers in eukaryotic signal transduction pathways. Intracellular Ca2+ concentration are modulated by various signals such as hormones and biotic and abiotic stresses. Modulation of Ca2+ ion leads to stimulation of calcium dependent protein kinase genes (CPKs), which results in regulation of gene expression and therefore mediates plant growth and development as well as biotic and abiotic stresses. Here, we reported the CPK gene family of 40 different plant species (950 CPK genes) and provided a unified nomenclature system for all of them. In addition, we analyzed their genomic, biochemical and structural conserved features. Multiple sequence alignment revealed that the kinase domain, auto-inhibitory domain and EF-hands regions of regulatory domains are highly conserved in nature. Additionally, the EF-hand domains of higher plants were found to contain four D-x-D and two D-E-L motifs, while lower eukaryotic plants had two D-x-D and one D-x-E motifs in their EF-hands. Phylogenetic analysis showed that CPK genes are clustered into four different groups. By studying the CPK gene family across the plant lineage, we provide the first evidence of the presence of D-x-D motif in the calcium binding EF-hand domain of CPK proteins.

Genome-wide identification of Calcineurin B-Like (CBL) gene family of plants reveals novel conserved motifs and evolutionary aspects in calcium signaling events.

BackgroundCalcium ions, the most versatile secondary messenger found in plants, are involved in the regulation of diverse arrays of plant growth and development, as well as biotic and abiotic stress responses. The calcineurin B-like proteins are one of the most important genes that act as calcium sensors.ResultsIn this study, we identified calcineurin B-like gene family members from 38 different plant species and assigned a unique nomenclature to each of them. Sequence analysis showed that, the CBL proteins contain three calcium binding EF-hand domain that contains several conserved Asp and Glu amino acid residues. The third EF-hand of the CBL protein was found to posses the D/E-x-D calcium binding sensor motif. Phylogenetic analysis showed that, the CBL genes fall into six different groups. Additionally, except group B CBLs, all the CBL proteins were found to contain N-terminal palmitoylation and myristoylation sites. An evolutionary study showed that, CBL genes are evolved from a common ancestor and subsequently diverged during the course of evolution of land plants. Tajima’s neutrality test showed that, CBL genes are highly polymorphic and evolved via decreasing population size due to balanced selection. Differential expression analysis with cold and heat stress treatment led to differential modulation of OsCBL genes.ConclusionsThe basic architecture of plant CBL genes is conserved throughout the plant kingdom. Evolutionary analysis showed that, these genes are evolved from a common ancestor of lower eukaryotic plant lineage and led to broadening of the calcium signaling events in higher eukaryotic organisms.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0543-0) contains supplementary material, which is available to authorized users.

The nphp-2 and arl-13 genetic modules interact to regulate ciliogenesis and ciliary microtubule patterning in C. elegans.

Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm C. elegans, the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that C. elegans cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the C. elegans ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. nphp-2 (the C. elegans Inversin homolog) and the doublet region genes arl-13, klp-11, and unc-119 are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—nphp-2+klp-11 and arl-13+unc-119—which are both antagonized by the hdac-6 deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by nphp-2, arl-13, and unc-119. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the C. elegans InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.

Calcium/Calmodulin-Dependent Protein Kinase Is Negatively and Positively Regulated by Calcium, Providing a Mechanism for Decoding Calcium Responses during Symbiosis Signaling

The establishment of symbiotic associations in plants requires calcium oscillations that must be decoded to invoke downstream developmental programs. In animal systems, comparable calcium oscillations are decoded by calmodulin (CaM)-dependent protein kinases, but symbiotic signaling involves a calcium/CaM-dependent protein kinase (CCaMK) that is unique to plants. CCaMK differs from the animal CaM kinases by its dual ability to bind free calcium, via calcium binding EF-hand domains on the protein, or to bind calcium complexed with CaM, via a CaM binding domain. In this study, we dissect this dual regulation of CCaMK by calcium. We find that calcium binding to the EF-hand domains promotes autophosphorylation, which negatively regulates CCaMK by stabilizing the inactive state of the protein. By contrast, calcium-dependent CaM binding overrides the effects of autophosphorylation and activates the protein. The differential calcium binding affinities of the EF-hand domains compared with those of CaM suggest that CCaMK is maintained in the inactive state at basal calcium concentrations and is activated via CaM binding during calcium oscillations. This work provides a model for decoding calcium oscillations that uses differential calcium binding affinities to create a robust molecular switch that is responsive to calcium concentrations associated with both the basal state and with oscillations.

Calcium-induced Conformational Changes in C-terminal Tail of Polycystin-2 Are Necessary for Channel Gating

Polycystin-2 (PC2) is a Ca(2+)-permeable transient receptor potential channel activated and regulated by changes in cytoplasmic Ca(2+). PC2 mutations are responsible for ∼15% of autosomal dominant polycystic kidney disease. Although the C-terminal cytoplasmic tail of PC2 has been shown to contain a Ca(2+)-binding EF-hand domain, the molecular basis of PC2 channel gating by Ca(2+) remains unknown. We propose that the PC2 EF-hand is a Ca(2+) sensor required for channel gating. Consistent with this, Ca(2+) binding causes a dramatic decrease in the radius of gyration (R(g)) of the PC2 EF-hand by small angle x-ray scattering and significant conformational changes by NMR. Furthermore, increasing Ca(2+) concentrations cause the C-terminal cytoplasmic tail to transition from a mixture of extended oligomers to a single compact dimer by analytical ultracentrifugation, coupled with a >30 Å decrease in maximum interatomic distance (D(max)) by small angle x-ray scattering. Mutant PC2 channels unable to bind Ca(2+) via the EF-hand are inactive in single-channel planar lipid bilayers and inhibit Ca(2+) release from ER stores upon overexpression in cells, suggesting dominant negative properties. Our results support a model where PC2 channels are gated by discrete conformational changes in the C-terminal cytoplasmic tail in response to changes in cytoplasmic Ca(2+) levels. These properties of PC2 are lost in autosomal dominant polycystic kidney disease, emphasizing the importance of PC2 to kidney cell function. We speculate that PC2 and the Ca(2+)-dependent transient receptor potential channels in general are regulated by similar conformational changes in their cytoplasmic domains that are propagated to the channel pore.

Cytosolic Calcium Dependency of STIM1 De-Oligomerization

Store operated calcium entry (SOCE) is a universal mechanism that cells use to replenish their intracellular calcium stores. SOCE activation is regulated by STIM1, an endoplasmic reticulum (ER) transmembrane protein with calcium binding EF-hand domains protruding into the ER lumen. Upon depletion of ER calcium stores, the dissociation of calcium ions from STIM1 EF-hands triggers a conformational change that leads to the formation of STIM1 oligomers, which translocate to the plasma membrane to bind and activate calcium channels of the Orai family.

Endoplasmic reticulum stress or mutation of an EF-hand Ca2+-binding domain directs the FKBP65 rotamase to an ERAD-based proteolysis

FKBP65 is an endoplasmic reticulum (ER)-localized chaperone and rotamase, with cargo proteins that include tropoelastin and collagen. In humans, mutations in FKBP65 have recently been shown to cause a form of osteogenesis imperfecta (OI), a brittle bone disease resulting from deficient secretion of mature type I collagen. In this work, we describe the rapid proteolysis of FKBP65 in response to ER stress signals that activate the release of ER Ca(2+) stores. A large-scale screen for stress-induced cellular changes revealed FKBP65 proteins to decrease within 6-12h of stress activation. Inhibiting IP(3)R-mediated ER Ca(2+) release blocked this response. No other ER-localized chaperone and folding mediators assessed in the study displayed this phenomenon, indicating that this rapid proteolysis of folding mediator is distinctive. Imaging and cellular fractionation confirmed the localization of FKBP65 (72kDa glycoprotein) to the ER of untreated cells, a rapid decrease in protein levels following ER stress, and the corresponding appearance of a 30-kDa fragment in the cytosol. Inhibition of the proteasome during ER stress revealed an accumulation of FKBP65 in the cytosol, consistent with retrotranslocation and a proteasome-based proteolysis. To assess the role of Ca(2+)-binding EF-hand domains in FKBP65 stability, a recombinant FKBP65-GFP construct was engineered to ablate Ca(2+) binding at each of two EF-hand domains. Cells transfected with the wild-type construct displayed ER localization of the FKBP65-GFP protein and a proteasome-dependent proteolysis in response to ER stress. Recombinant FKBP65-GFP carrying a defect in the EF1 Ca(2+)-binding domain displayed diminished protein in the ER when compared to wild-type FKBP65-GFP. Proteasome inhibition restored mutant protein to levels similar to that of the wild-type FKBP65-GFP. A similar mutation in EF2 did not confer FKBP65 proteolysis. This work supports a model in which stress-induced changes in ER Ca(2+) stores induce the rapid proteolysis of FKBP65, a chaperone and folding mediator of collagen and tropoelastin. The destruction of this protein may identify a cellular strategy for replacement of protein folding machinery following ER stress. The implications for stress-induced changes in the handling of aggregate-prone proteins in the ER-Golgi secretory pathway are discussed. This work was supported by grants from the National Institutes of Health (R15GM065139) and the National Science Foundation (DBI-0452587).

Critical Role of Calcium in the Regulation of the ER-to-Golgi Transport of FV and FVIII by the LMAN1-MCFD2 Cargo Receptor.

Abstract 2140Poster Board II-117Coagulation factor V (FV) and factor VIII (FVIII) play key roles in hemostasis and thrombosis. The LMAN1 (ERGIC-53)-MCFD2 complex is a mammalian cargo receptor for efficient transport of FV and FVIII from the endoplasmic reticulum (ER) to the Golgi. Mutations in either LMAN1 or MCFD2 cause a bleeding disorder, combined deficiency of factor V and factor VIII. LMAN1 is a type-1 transmembrane protein with a Ca2+-dependent carbohydrate recognition domain homologous to leguminous lectins. MCFD2 is a small soluble protein with an N-terminal sequence of unknown structure and two Ca2+-binding EF-hand domains at the C terminus. LMAN1 and MCFD2 form a Ca2+-dependent protein complex in the ER-Golgi intermediate compartment (ERGIC), an organelle between the ER and Golgi that is unique to higher eukaryotic cells. FV and FVIII interact with the LMAN1-MCFD2 complex in a Ca2+ -dependent manner. To elucidate the role of Ca2+ in regulating the ER-to-Golgi transport of FV and FVIII, we determined the structural features important for the organization of the receptor complex and the interaction of this complex with its client cargo FV and FVIII. We show that the C-terminal Ca2+-binding EF hand domains of MCFD2 are both necessary and sufficient for interaction with LMAN1. The EF hand domains also mediate the interaction with FV and FVIII. All MCFD2 missense mutants identified in F5F8D patients are localized to the EF hand domains and fail to bind LMAN1. However, these mutants still retain the FV and FVIII binding activities. Circular dichroism spectroscopy studies on missense mutations localized to different structural elements of the EF hand domains suggest that Ca2+-induced folding of MCFD2 is important for LMAN1 interaction, but not essential for FV and FVIII binding. We also demonstrate that the carbohydrate recognition domain (CRD) of LMAN1 contains separate binding sites for MCFD2 and FV/FVIII. Mutations in the Ca2+ and sugar binding sites of CRD disrupt FV and FVIII interaction, without affecting MCFD2 binding, suggesting that the Ca2+ binding sites in LMAN1 are primarily required for the recognition of sugar residues in FV and FVIII. These results support a model in which Ca2+ plays a critical role in regulating the binding in the ER and the subsequent release in the ERGIC of FV and FVIII. Ca2+ concentration is higher in the ER than in the ERGIC and the Golgi. In the ER lumen, FV and FVIII loading is initiated by a flexible interaction with MCFD2 and stabilized by the follow-up interaction of sugar side chains of FV and FVIII with the carbohydrate binding site of LMAN1. The LMAN1-FV/FVIII interaction is more sensitive to Ca2+ concentration than the LMAN1-MCFD2 interaction, so that the lower Ca2+/pH in the ERGIC triggers the release of FV and FVIII but not the dissociation of the LMAN1-MCFD2 receptor complex. The empty receptor complex is subsequently recycled back to the ER for the next round of cargo loading. Disclosures:No relevant conflicts of interest to declare.

The Short N-terminal Domains of STIM1 and STIM2 Control the Activation Kinetics of Orai1 Channels

STIM1 and STIM2 are dynamic transmembrane endoplasmic reticulum Ca(2+) sensors, coupling directly to activate plasma membrane Orai Ca(2+) entry channels. Despite extensive sequence homology, the STIM proteins are functionally distinct. We reveal that the short variable N-terminal random coil sequences of STIM1 and STIM2 confer profoundly different activation properties. Using Orai1-expressing HEK293 cells, chimeric replacement of the 43-amino-acid STIM1 N terminus with that of STIM2 attenuates Orai1-mediated Ca(2+) entry and drastically slows store-induced Orai1 channel activation. Conversely, the 55-amino-acid STIM2 terminus substituted within STIM1 strikingly enhances both Orai1-mediated Ca(2+) entry and constitutive coupling to activate Orai1 channels. Hence, STIM N termini are powerful coupling modifiers, functioning in STIM2 to "brake" the otherwise constitutive activation of Orai1 channels afforded by its high sensitivity to luminal Ca(2+).