Activation of the B cell receptor complex in B lymphocytes causes Ca2+ release from intracellular stores, which, in turn, activates ion channels known as Icrac. We investigated the mechanisms that link Ca2+ store release to channel gating in DT40 B lymphocyte cell lines genetically manipulated to suppress the expression of several tyrosine kinases: Btk, Lyn, Syk, and the Blnk adaptor molecule. The simultaneous but not the independent suppression of Lyn and Syk expression prevents the activation of Icrac without interfering with thapsigargin-sensitive Ca2+ store release. Icrac activation by Ca2+ is reversed in mutant cells by the homologous expression of the missing kinases. Pharmacological inhibition of kinase activity by LavendustinA and PP2 cause the same functional deficit as the genetic suppression of enzyme expression. Biochemical assays demonstrate that kinase activity is required as a tonic signal: targets must be phosphorylated to link Ca2+ store release to Icrac gating. The action of kinases on Icrac activation does not arise from control of the expression level of the stromal interaction molecule 1 and Orai1 proteins.
All eukaryotic cells have adapted the use of the calcium ion (Ca) as a universal signaling element through the evolution of a toolkit of Ca sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca can subsequently signal the opening surface channels which permit the movement of Ca from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca sinks by taking up Ca in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IPRs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca from the extracellular space to the cytosol; uptake of Ca into the matrix through the mitochondrial Ca uniporter (MCU) further shapes the cytosolic Ca levels. Recent structural elucidations of these key Ca toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IPR, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.
Stromal interaction molecule (STIM)-1 and -2 regulate agonist-induced and basal cytosolic calcium (Ca2+) levels after oligomerization and translocation to endoplasmic reticulum (ER)-plasma membrane (PM) junctions. At these junctions, the STIM cytosolic coiled-coil (CC) domains couple to PM Orai1 proteins and gate these Ca2+ release-activated Ca2+ (CRAC) channels, which facilitate store-operated Ca2+ entry (SOCE). Unlike STIM1 and STIM2, which are SOCE activators, the STIM2β splice variant contains an 8-residue insert located within the conserved CCs which inhibits SOCE. It remains unclear if the 2β insert further depotentiates weak STIM2 coupling to Orai1 or independently causes structural perturbations which prevent SOCE. Here, we use far-UV circular dichroism, light scattering, exposed hydrophobicity analysis, solution small angle X-ray scattering, and a chimeric STIM1/STIM2β functional assessment to provide insights into the molecular mechanism by which the 2β insert precludes SOCE activation. We find that the 2β insert reduces the overall α-helicity and enhances the exposed hydrophobicity of the STIM2 CC domains in the absence of a global conformational change. Remarkably, incorporation of the 2β insert into the STIM1 context not only affects the secondary structure and hydrophobicity as observed for STIM2, but also eliminates the more robust SOCE response mediated by STIM1. Collectively, our data show that the 2β insert directly precludes Orai1 channel activation by inducing structural perturbations in the STIM CC region.
To promote active transportation modes (such as bike ride and walking), and to create safer communities for easier access to transit, it is essential to provide consolidated data-driven transportation information to the public. The relevant and timely information from data facilitates the improvement of decision-making processes for the establishment of public policy and urban planning for sustainable growth, and for promoting public health in the region. For the characterization of the spatial variation of transportation-emitted air pollution in the Fresno/Clovis neighborhood in California, various species of particulate matters emitted from traffic sources were measured using real-time monitors and GPS loggers at over 100 neighborhood walking routes within 58 census tracts from the previous research, Children’s Health to Air Pollution Study - San Joaquin Valley (CHAPS-SJV). Roadside air pollution data show that PM2.5, black carbon, and PAHs were significantly elevated in the neighborhood walking air samples compared to indoor air or the ambient monitoring station in the Central Fresno area due to the immediate source proximity. The simultaneous parallel measurements in two neighborhoods which are distinctively different areas (High diesel High poverty vs. Low diesel Low poverty) showed that the higher pollution levels were observed when more frequent vehicular activities were occurring around the neighborhoods. Elevated PM2.5 concentrations near the roadways were evident with a high volume of traffic and in regions with more unpaved areas. Neighborhood walking air samples were influenced by immediate roadway traffic conditions, such as encounters with diesel trucks, approaching in close proximity to freeways and/or busy roadways, passing cigarette smokers, and gardening activity. The elevated black carbon concentrations occur near the highway corridors and regions with high diesel traffic and high industry. This project provides consolidated data-driven transportation information to the public including: 1. Transportation-related particle pollution data 2. Spatial analyses of geocoded vehicle emissions 3. Neighborhood characterization for the built environment such as cities, buildings, roads, parks, walkways, etc.
The San Joaquin Valley is identified as an area with a high level of particulate matter (PM) in the air, reaching above the federal and state clean air standards (EPA 2019). Many of the cities in the valley are classified as the most polluted cities in the United States for both particulate matter and ozone pollution (American Lung Association, 2021). To resolve this issue, alternative forms of transportation have been considered in transportation planning. In this study, active transportation mode air quality was monitored on selected Woodward Park and Old Clovis trails and urban bike lanes. Real-time aerosol monitors, and low-cost sensors were carried in a backpack on bicycles during the sampling. Researchers collected GPS data via a portable GPS technology called Tracksticks. Driving transportation mode air quality data was acquired from the roadways within the Fresno/Clovis area, spanning six sampling routes, and during intercity trips between Fresno, Berkeley, and Los Angeles, for a total of five sampling routes. ‘On-Road' (outside vehicle) monitors were installed on the roof of a vehicle while ‘In-Vehicle’ monitors were installed inside the vehicle for comparison with the particulate pollution levels in the two contrasting microenvironments. The results showed the following three main outcomes: (1) clear relationships exist among PMs of different sizes; (2) there were greater variations in air quality of bike trails and On-Road samples than backyard and In-Vehicle samples; (3) we observed significant differences in air quality inside and outside the vehicle while driving local and intercity roadways; and (4) the road trip to the Bay area revealed that San Joaquin Valley has increased ambient PM2.5 and black carbon (BC) levels compared to those in the Bay Area on every trip, regardless of the daily change of the air quality.
Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca) entry which is the principal Ca signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.
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