Temporal datalog with existential quantification

Existential rules, also known as tuple-generating dependencies (TGDs) or Datalog± rules, are heavily studied in the communities of Knowledge Representation and Reasoning, Semantic Web, and Databases, due to their rich modelling capabilities. In this paper we consider TGDs in the temporal setting, by introducing and studying DatalogMTL∃—an extension of metric temporal Datalog (DatalogMTL) obtained by allowing for existential rules in programs. We show that DatalogMTL∃ is undecidable even in the restricted cases of guarded and weakly-acyclic programs. To address this issue we introduce uniform semantics which, on the one hand, is well-suited for modelling temporal knowledge as it prevents from unintended value invention and, on the other hand, provides decidability of reasoning; in particular, it becomes 2-ExpSpace-complete for weakly-acyclic programs but remains undecidable for guarded programs. We provide an implementation for the decidable case and demonstrate its practical feasibility. Thus we obtain an expressive, yet decidable, rule-language and a system which is suitable for complex temporal reasoning with existential rules

Discovery of novel gating checkpoints in the Orai1 calcium channel by systematic analysis of constitutively active mutants of its paralogs and orthologs.

In humans, there are three paralogs of the Orai Ca2+ channel that form the core of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still under active investigation, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating checkpoints among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the "ANSGA" nexus mutations in TM4 of human Orai1, which is reported to mimic the STIM1-activated state of the channel. In investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, that are not conserved among paralogs and that seem to be crucial for the channel activation triggered by the "ANSGA" mutations in Orai1. However, mutations of the same residues still allow gating of Orai1 by STIM1, suggesting that the ANSGA mutant of Orai1 may not be a surrogate for the STIM1-activated state of the Orai1 channel. Our results shed new light on these important gating checkpoints and show that the gating mechanism of Orai channels is affected by multiple factors that are not necessarily conserved among orai homologs, such as the TM4-TM3 coupling

Characterisation and structural investigation of disease-related Orai1-T184 mutants

Cations, especially calcium (Ca2+), play important roles in many biological processes. Gene regulation, immune defence as well as cell growth and death are initiated and regulated by the entry and release of Ca2+ in excitable and non-excitable cells. The calcium balance is regulated through protein-systems, which are responsible for the regulated reception and delivery of Ca2+. The actual Ca2+ level of calcium stores within eukaryotic cells, mainly the endoplasmic/sarcoplasmic reticulum (ER/SR), plays a key role during store-operated Ca2+ entry (SOCE) signal propagation. The depletion of Ca2+-stores in different cell types activates signalling pathways, which lead to an entry of Ca2+ through store-operated calcium channels (SOCC) from outside the cells into the cytosol, activating different Ca2+ depending pathways, followed by the refilling of the calcium stores. The most prominent SOCC is the so called Ca2+ release activated Ca2+ (CRAC) channel. This channel is composed of two key players called stromal interaction molecule 1 (STIM1) and Orai1. STIM1 acts as the Ca2+ sensor located in the ER-membrane whereas Orai1 is located in the plasma membrane, and is known to be the pore forming unit of the CRAC channel. The interaction between these two proteins upon ER-store depletion leads to Ca2+ influx via CRAC channels that represent a main signalling pathway for a variety of cellular functions. During years of research several disease-related mutations of STIM1 and Orai1 proteins were identified. The differentiation of muscle cells is regulated by Ca2+ influx through STIM1-Orai1 interactions. A skeletal muscle disease called tubular aggregate myopathy (TAM), which causes muscle pain, cramping or weakens was associated with Orai1 and STIM1 mutations. In this work a single point mutation T184M, located in the third transmembrane helix of the Orai1 protein was investigated. This mutation among others, is assumed to prevent the approachability of reactive oxygen species (ROS) to specific cysteines in transmembrane helix 3 of Orai1 and therefore block the negative modulation of the Orai1 function by ROS. Within this thesis, effects onto the Orai1-protein structure of different T184 mutations were investigated via cysteine cross-linking experiments. Furthermore patch-clamp, protein localisation as well as NFAT experiments were conducted to further understand the influence of the different mutations of the T184 amino acid residue on the Orai1 protein structure, activation/gating process as wells as on the Ca2+ signal cascade.submitted by Matthias Sallinger BSc.Universität Linz, Masterarbeit, 2020(VLID)470639

More Than Just Simple Interaction between STIM and Orai Proteins: CRAC Channel Function Enabled by a Network of Interactions with Regulatory Proteins

The calcium-release-activated calcium (CRAC) channel, activated by the release of Ca2+ from the endoplasmic reticulum (ER), is critical for Ca2+ homeostasis and active signal transduction in a plethora of cell types. Spurred by the long-sought decryption of the molecular nature of the CRAC channel, considerable scientific effort has been devoted to gaining insights into functional and structural mechanisms underlying this signalling cascade. Key players in CRAC channel function are the Stromal interaction molecule 1 (STIM1) and Orai1. STIM1 proteins span through the membrane of the ER, are competent in sensing luminal Ca2+ concentration, and in turn, are responsible for relaying the signal of Ca2+ store-depletion to pore-forming Orai1 proteins in the plasma membrane. A direct interaction of STIM1 and Orai1 allows for the re-entry of Ca2+ from the extracellular space. Although much is already known about the structure, function, and interaction of STIM1 and Orai1, there is growing evidence that CRAC under physiological conditions is dependent on additional proteins to function properly. Several auxiliary proteins have been shown to regulate CRAC channel activity by means of direct interactions with STIM1 and/or Orai1, promoting or hindering Ca2+ influx in a mechanistically diverse manner. Various proteins have also been identified to exert a modulatory role on the CRAC signalling cascade although inherently lacking an affinity for both STIM1 and Orai1. Apart from ubiquitously expressed representatives, a subset of such regulatory mechanisms seems to allow for a cell-type-specific control of CRAC channel function, considering the rather restricted expression patterns of the specific proteins. Given the high functional and clinical relevance of both generic and cell-type-specific interacting networks, the following review shall provide a comprehensive summary of regulators of the multilayered CRAC channel signalling cascade. It also includes proteins expressed in a narrow spectrum of cells and tissues that are often disregarded in other reviews of similar topics

Calcium Signals during SARS-CoV-2 Infection: Assessing the Potential of Emerging Therapies

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a positive-sense single-stranded RNA virus that causes coronavirus disease 2019 (COVID-19). This respiratory illness was declared a pandemic by the world health organization (WHO) in March 2020, just a few weeks after being described for the first time. Since then, global research effort has considerably increased humanity’s knowledge about both viruses and disease. It has also spawned several vaccines that have proven to be key tools in attenuating the spread of the pandemic and severity of COVID-19. However, with vaccine-related skepticism being on the rise, as well as breakthrough infections in the vaccinated population and the threat of a complete immune escape variant, alternative strategies in the fight against SARS-CoV-2 are urgently required. Calcium signals have long been known to play an essential role in infection with diverse viruses and thus constitute a promising avenue for further research on therapeutic strategies. In this review, we introduce the pivotal role of calcium signaling in viral infection cascades. Based on this, we discuss prospective calcium-related treatment targets and strategies for the cure of COVID-19 that exploit viral dependence on calcium signals

Science CommuniCa2+tion Developing Scientific Literacy on Calcium: The Involvement of CRAC Currents in Human Health and Disease

All human life starts with a calcium (Ca2+) wave. This ion regulates a plethora of cellular functions ranging from fertilisation and birth to development and cell death. A sophisticated system is responsible for maintaining the essential, tight concentration of calcium within cells. Intricate components of this Ca2+ network are store-operated calcium channels in the cells’ membrane. The best-characterised store-operated channel is the Ca2+ release-activated Ca2+ (CRAC) channel. Currents through CRAC channels are critically dependent on the correct function of two proteins: STIM1 and Orai1. A disruption of the precise mechanism of Ca2+ entry through CRAC channels can lead to defects and in turn to severe impacts on our health. Mutations in either STIM1 or Orai1 proteins can have consequences on our immune cells, the cardiac and nervous system, the hormonal balance, muscle function, and many more. There is solid evidence that altered Ca2+ signalling through CRAC channels is involved in the hallmarks of cancer development: uncontrolled cell growth, resistance to cell death, migration, invasion, and metastasis. In this work we highlight the importance of Ca2+ and its role in human health and disease with focus on CRAC channels

Luminal STIM1 Mutants that Cause Tubular Aggregate Myopathy Promote Autophagic Processes

Stromal interaction molecule 1 (STIM1) is a ubiquitously expressed Ca2+ sensor protein that induces permeation of Orai Ca2+ channels upon endoplasmic reticulum Ca2+-store depletion. A drop in luminal Ca2+ causes partial unfolding of the N-terminal STIM1 domains and thus initial STIM1 activation. We compared the STIM1 structure upon Ca2+ depletion from our molecular dynamics (MD) simulations with a recent 2D NMR structure. Simulation- and structure-based results showed unfolding of two α-helices in the canonical and in the non-canonical EF-hand. Further, we structurally and functionally evaluated mutations in the non-canonical EF-hand that have been shown to cause tubular aggregate myopathy. We found these mutations to cause full constitutive activation of Ca2+-release-activated Ca2+ currents (ICRAC) and to promote autophagic processes. Specifically, heterologously expressed STIM1 mutations in the non-canonical EF-hand promoted translocation of the autophagy transcription factors microphthalmia-associated transcription factor (MITF) and transcription factor EB (TFEB) into the nucleus. These STIM1 mutations additionally stimulated an enhanced production of autophagosomes. In summary, mutations in STIM1 that cause structural unfolding promoted Ca2+ down-stream activation of autophagic processes

Orai1 Boosts SK3 Channel Activation

The interplay of SK3, a Ca2+ sensitive K+ ion channel, with Orai1, a Ca2+ ion channel, has been reported to increase cytosolic Ca2+ levels, thereby triggering proliferation of breast and colon cancer cells, although a molecular mechanism has remained elusive to date. We show in the current study, via heterologous protein expression, that Orai1 can enhance SK3 K+ currents, in addition to constitutively bound calmodulin (CaM). At low cytosolic Ca2+ levels that decrease SK3 K+ permeation, co-expressed Orai1 potentiates SK3 currents. This positive feedback mechanism of SK3 and Orai1 is enabled by their close co-localization. Remarkably, we discovered that loss of SK3 channel activity due to overexpressed CaM mutants could be restored by Orai1, likely via its interplay with the SK3–CaM binding site. Mapping for interaction sites within Orai1, we identified that the cytosolic strands and pore residues are critical for a functional communication with SK3. Moreover, STIM1 has a bimodal role in SK3–Orai1 regulation. Under physiological ionic conditions, STIM1 is able to impede SK3–Orai1 interplay by significantly decreasing their co-localization. Forced STIM1–Orai1 activity and associated Ca2+ influx promote SK3 K+ currents. The dynamic regulation of Orai1 to boost endogenous SK3 channels was also determined in the human prostate cancer cell line LNCaP

Dissecting gating mechanisms of Orai calcium channel paralogs using constitutively active Orai mutants that mimic STIM1-gated state

In humans, there are three paralogs of the Orai Ca2+ channel, which lie at the heart of the store-operated calcium entry (SOCE) machinery. While the STIM-mediated gating mechanism of Orai channels is still being actively investigated, several artificial and natural variants are known to cause constitutive activity of the human Orai1 channel. Surprisingly, little is known about the conservation of the gating mechanism among the different human Orai paralogs and orthologs in other species. In our work, we show that the mutation corresponding to the activating mutation H134A in transmembrane helix 2 (TM2) of human Orai1 also activates Orai2 and Orai3, likely via a similar mechanism. However, this cross-paralog conservation does not apply to the “ANSGA” nexus mutations in TM4 of human Orai1 which mimic the STIM1-activated state of the channel. Investigating the mechanistic background of these differences, we identified two positions, H171 and F246 in human Orai1, which directly control the channel activation triggered by the “ANSGA” mutations in Orai1. Our results shed new light on these important gating checkpoints and show that the gating mechanism of the Orai channels is affected by multiple factors that are not necessarily evolutionarily conserved, such as the TM4-TM3 coupling

Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker