Sviatoslav N. Bagriantsev

PI(4,5)P2-Dependent and Ca2+-Regulated ER-PM Interactions Mediated by the Extended Synaptotagmins

Most available information on endoplasmic reticulum (ER)-plasma membrane (PM) contacts in cells of higher eukaryotes concerns proteins implicated in the regulation of Ca(2+) entry. However, growing evidence suggests that such contacts play more general roles in cell physiology, pointing to the existence of additionally ubiquitously expressed ER-PM tethers. Here, we show that the three extended synaptotagmins (E-Syts) are ER proteins that participate in such tethering function via C2 domain-dependent interactions with the PM that require PI(4,5)P2 in the case of E-Syt2 and E-Syt3 and also elevation of cytosolic Ca(2+) in the case of E-Syt1. As they form heteromeric complexes, the E-Syts confer cytosolic Ca(2+) regulation to ER-PM contact formation. E-Syts-dependent contacts, however, are not required for store-operated Ca(2+) entry. Thus, the ER-PM tethering function of the E-Syts (tricalbins in yeast) mediates the formation of ER-PM contacts sites, which are functionally distinct from those mediated by STIM1 and Orai1.

Analysis of Amyloid Aggregates Using Agarose Gel Electrophoresis

Amyloid aggregates are associated with a number of mammalian neurodegenerative diseases. Infectious aggregates of the mammalian prion protein PrP(sc) are hallmarks of transmissible spongiform encephalopathies in humans and cattle (Griffith, 1967; Legname et al., 2004; Prusiner, 1982; Silveira et al., 2004). Likewise, SDS-stable aggregates and low-n oligomers of the Abeta peptide (Selkoe et al., 1982; Walsh et al., 2002) cause toxic effects associated with Alzheimers disease (Selkoe, 2004). The discovery of prions in lower eukaryotes, for example, yeast prions [PSI(+)], [PIN(+)], and [URE3] suggested that prion phenomena may represent a fundamental process that is widespread among living organisms (Chernoff, 2004; Uptain and Lindquist, 2002; Wickner, 1994; Wickner et al., 2004). These protein structures are more stable than other cellular protein complexes, which generally dissolve in SDS at room temperature. In contrast, the prion polymers withstand these conditions, while losing their association with their non-prion partners. These bulky protein particles cannot be analyzed in polyacrylamide gels, because their pores are too small to allow the passage and acceptable resolution of the large complexes. This problem was first circumvented by Kryndushkin et al. (2003), who used Western blots of protein complexes separated on agarose gels to analyze the sizes of SDS-resistant protein complexes associated with the yeast prion [PSI(+)]. Further studies have used this approach to characterize [PSI(+)] (Allen et al., 2005; Bagriantsev and Liebman, 2004; Salnikova et al., 2005), and another yeast prion [PIN(+)] (Bagriantsev and Liebman, 2004). In this chapter, we use this method to assay amyloid aggregates of recombinant proteins Sup35NM and Abeta42 and present protocols for Western blot analysis of high molecular weight (>5 MDa) amyloid aggregates resolved in agarose gels. The technique is suitable for the analysis of any large proteins or SDS-stable high molecular weight complexes.

Specificity of prion assembly in vivo ; [PSI^+] and [PIN^+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion “strains,” [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.

Modulation of Aβ42 low-n oligomerization using a novel yeast reporter system

BackgroundWhile traditional models of Alzheimers disease focused on large fibrillar deposits of the Aβ42 amyloid peptide in the brain, recent work suggests that the major pathogenic effects may be attributed to SDS-stable oligomers of Aβ42. These Aβ42 oligomers represent a rational target for therapeutic intervention, yet factors governing their assembly are poorly understood.ResultsWe describe a new yeast model system focused on the initial stages of Aβ42 oligomerization. We show that the activity of a fusion of Aβ42 to a reporter protein is compromised in yeast by the formation of SDS-stable low-n oligomers. These oligomers are reminiscent of the low-n oligomers formed by the Aβ42 peptide in vitro, in mammalian cell culture, and in the human brain. Point mutations previously shown to inhibit Aβ42 aggregation in vitro, were made in the Aβ42 portion of the fusion protein. These mutations both inhibited oligomerization and restored activity to the fusion protein. Using this model system, we found that oligomerization of the fusion protein is stimulated by millimolar concentrations of the yeast prion curing agent guanidine. Surprisingly, deletion of the chaperone Hsp104 (a known target for guanidine) inhibited oligomerization of the fusion protein. Furthermore, we demonstrate that Hsp104 interacts with the Aβ42-fusion protein and appears to protect it from disaggregation and degradation.ConclusionPrevious models of Alzheimers disease focused on unravelling compounds that inhibit fibrillization of Aβ42, i.e. the last step of Aβ42 assembly. However, inhibition of fibrillization may lead to the accumulation of toxic oligomers of Aβ42. The model described here can be used to search for and test proteinacious or chemical compounds for their ability to interfere with the initial steps of Aβ42 oligomerization. Our findings suggest that yeast contain guanidine-sensitive factor(s) that reduce the amount of low-n oligomers of Aβ42. As many yeast proteins have human homologs, identification of these factors may help to uncover homologous proteins that affect Aβ42 oligomerization in mammals.

Multiple modalities converge on a common gate to control K2P channel function

Members of the K2P potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K2Ps are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K2P, K2P2.1 (KCNK2/TREK‐1), at a common molecular gate that comprises elements of the pore‐forming segments and the N‐terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K2Ps and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K2P family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus.

Piezo Proteins: Regulators of Mechanosensation and Other Cellular Processes

Piezo proteins have recently been identified as ion channels mediating mechanosensory transduction in mammalian cells. Characterization of these channels has yielded important insights into mechanisms of somatosensation, as well as other mechano-associated biologic processes such as sensing of shear stress, particularly in the vasculature, and regulation of urine flow and bladder distention. Other roles for Piezo proteins have emerged, some unexpected, including participation in cellular development, volume regulation, cellular migration, proliferation, and elongation. Mutations in human Piezo proteins have been associated with a variety of disorders including hereditary xerocytosis and several syndromes with muscular contracture as a prominent feature.

Metabolic and thermal stimuli control K 2P 2.1 (TREK-1) through modular sensory and gating domains

K2P2.1 (TREK‐1) is a polymodal two‐pore domain leak potassium channel that responds to external pH, GPCR‐mediated phosphorylation signals, and temperature through the action of distinct sensors within the channel. How the various intracellular and extracellular sensory elements control channel function remains unresolved. Here, we show that the K2P2.1 (TREK‐1) intracellular C‐terminal tail (Ct), a major sensory element of the channel, perceives metabolic and thermal commands and relays them to the extracellular C‐type gate through transmembrane helix M4 and pore helix 1. By decoupling Ct from the pore‐forming core, we further demonstrate that Ct is the primary heat‐sensing element of the channel, whereas, in contrast, the pore domain lacks robust temperature sensitivity. Together, our findings outline a mechanism for signal transduction within K2P2.1 (TREK‐1) in which there is a clear crosstalk between the C‐type gate and intracellular Ct domain. In addition, our findings support the general notion of the existence of modular temperature‐sensing domains in temperature‐sensitive ion channels. This marked distinction between gating and sensory elements suggests a general design principle that may underlie the function of a variety of temperature‐sensitive channels.

A high-throughput functional screen identifies small molecule regulators of temperature- and mechano-sensitive K2P channels

K2P (KCNK) potassium channels generate “leak” potassium currents that strongly influence cellular excitability and contribute to pain, somatosensation, anesthesia, and mood. Despite their physiological importance, K2Ps lack specific pharmacology. Addressing this issue has been complicated by the challenges that the leak nature of K2P currents poses for electrophysiology-based high-throughput screening strategies. Here, we present a yeast-based high-throughput screening assay that avoids this problem. Using a simple growth-based functional readout, we screened a library of 106,281 small molecules and identified two new inhibitors and three new activators of the mammalian K2P channel K2P2.1 (KCNK2, TREK-1). By combining biophysical, structure–activity, and mechanistic analysis, we developed a dihydroacridine analogue, ML67-33, that acts as a low micromolar, selective activator of temperature- and mechano-sensitive K2P channels. Biophysical studies show that ML67-33 reversibly increases channel currents by activating the extracellular selectivity filter-based C-type gate that forms the core gating apparatus on which a variety of diverse modulatory inputs converge. The new K2P modulators presented here, together with the yeast-based assay, should enable both mechanistic and physiological studies of K2P activity and facilitate the discovery and development of other K2P small molecule modulators.

Guanidine reduces stop codon read-through caused by missense mutations in SUP35 or SUP45

Sup35 and Sup45 are essential protein components of the Saccharomyces cerevisiae translation termination factor. Yeast cells harbouring the [PSI+] prion form of Sup35 have impaired stop codon recognition (nonsense suppression). It has long been known that the [PSI+] prion is not stably transmitted to daughter cells when yeast are grown in the presence of mM concentrations of guanidine hydrochloride (GuHCl). In this paper, Mendelian suppressor mutations whose phenotypes are likewise hidden during growth in the presence of millimolar GuHCl are described. Such GuHCl‐remedial Mendelian suppressors were selected under conditions where [PSI+] appearance was limiting, and were caused by missense mutations in SUP35 or SUP45. Clearly, antisuppression caused by growth in the presence of GuHCl is not sufficient to distinguish missense mutations in SUP35 or SUP45, from [PSI+]. However, the Mendelian and prion suppressors can be distinguished by subsequent growth in the absence of GuHCl, where only the nonsense suppression caused by the [PSI+] prion remains cured. Recent reports indicate that GuHCl blocks the inheritance of [PSI+] by directly inhibiting the activity of the protein remodelling factor Hsp104, which is required for the transmission of [PSI+] from mother to daughter cells. However, the nonsense suppressor activity caused by the GuHCl‐remedial sup35 or sup45 suppressors does not require Hsp104. Thus, GuHCl must antisuppress the sup35 and sup45 mutations via an in vivo target distinct from Hsp104. Copyright

Neuronal mechanism for acute mechanosensitivity in tactile-foraging waterfowl

Significance Like vision, audition, and olfaction, mechanosensation is a fundamental way in which animals interact with the environment, but it remains the least well understood at the cellular and molecular levels. Here, we explored evolutionary changes that contribute to the enhancement of mechanosensitivity in tactile-foraging ducks. We found that the somatosensory neurons that innervate the duck bill can detect physical force much more efficiently than analogous cells in other species, such as mice. Furthermore, ducks exhibit an increase in the number of neurons dedicated to this task in their sensory ganglia and a decrease in the number of neurons that detect temperature. Our findings provide an explanation for the acute mechanosensitivity of the duck bill at the level of somatosensory neurons. Relying almost exclusively on their acute sense of touch, tactile-foraging birds can feed in murky water, but the cellular mechanism is unknown. Mechanical stimuli activate specialized cutaneous end organs in the bill, innervated by trigeminal afferents. We report that trigeminal ganglia (TG) of domestic and wild tactile-foraging ducks exhibit numerical expansion of large-diameter mechanoreceptive neurons expressing the mechano-gated ion channel Piezo2. These features are not found in visually foraging birds. Moreover, in the duck, the expansion of mechanoreceptors occurs at the expense of thermosensors. Direct mechanical stimulation of duck TG neurons evokes high-amplitude depolarizing current with a low threshold of activation, high signal amplification gain, and slow kinetics of inactivation. Together, these factors contribute to efficient conversion of light mechanical stimuli into neuronal excitation. Our results reveal an evolutionary strategy to hone tactile perception in vertebrates at the level of primary afferents.