Corrigendum: Increased Permeability of the Aquaporin SoPIP2;1 by Mercury and Mutations in Loop A

The publication of Andreas Cellarius\u27s Harmonia Macrocosmica in 1660 represented the completion of an ambitious cartographic project begun over twenty years earlier by the family of Johannes Jansonnius. Jansonnius had proposed to include in his multi-volume Novus Atlas a description of the whole world, that is \u27the Heavens and the Earth\u27. The series incorporated the famous Blaeu Atlas. Cellarius used elaborate illustrations to depict not only the Copernican \u27world system\u27 (model of the universe), but also the classical inheritance, Ptolemy\u27s geocentric model. The work became extremely popular and was frequently reprinted

Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

Background: SoPIP2;1 constitutes one of the major integral proteins in spinach leaf plasma membranes and belongs to the aquaporin family. SoPIP2;1 is a highly permeable and selective water channel that has been successfully overexpressed and purified with high yields. In order to optimize reconstitution of the purified protein into biomimetic systems, we have here for the first time characterized the structural stability of SoPIP2;1. Methodology/Principal Finding: We have characterized the protein structural stability after purification and after reconstitution into detergent micelles and proteoliposomes using circular dichroism and fluorescence spectroscopy techniques. The structure of SoPIP2;1 was analyzed either with the protein solubilized with octyl-beta-D-glucopyranoside (OG) or reconstituted into lipid membranes formed by E. coli lipids, diphytanoylphosphatidylcholine (DPhPC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a-helical in accordance with crystallographic data. The protein has a high thermal structural stability in detergent solutions, with an irreversible thermal unfolding occurring at a melting temperature of 58 degrees C. Incorporation of the protein into lipid membranes increases the structural stability as evidenced by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes for biotechnological applications

Characterization of Membrane Proteins: From a gated plant aquaporin to animal ion channel receptors

Membrane proteins play several important roles in a cell. Among these proteins are aquaporins (AQPs) and transient receptor potential (TRP) ion channels that mediate water transport, temperature and noxious chemical sensation, respectively. The function of some AQPs, for example the spinach isoform SoPIP2;1 is regulated by pH, phosphorylation and heavy metals such as mercury. However, the mechanisms by which mercury activate or inhibits AQPs are poorly understood. We suggest that mercury binds to SoPIP2;1 close to the C-terminal end and that the binding of mercury results in destabilization of the C-terminal region. This may affect its interaction with the residues forming the gate and therefore lead to an increase of the water permeability of SoPIP2;1 (Paper II). SoPIP2;1 is a highly selective water channel and can be produced as a functional protein in high yield in a heterologous system which suggest that SoPIP2;1 is a good choice for insertion in biomimetic membranes to be used for water purification. However, the stability of SoPIP2;1 in artificial membranes needed to be demonstrated. Thus we determined the stability of SoPIP2;1 in different lipids and identified E. coli polar lipids as the best system for reconstitution of SoPIP2;1. The results will contribute towards the effort to use SoPIP2;1 in biomimetic water filtration technology (Paper I). The animal TRP ion channel subtype A1 (TRPA1) from fruit fly, snake and mosquito has been implicated in warm temperature sensation. However, the threshold temperature which activates human TRPA1 (hTRPA1) is controversial. We addressed this issue by reconstituting the purified hTRPA1 in artificial lipid membranes. The purified hTRPA1 was found to be activated by cold temperatures and electrophilic chemicals. The results resolve the controversy surrounding the threshold temperature for the activation of hTRPA1 (Paper IV). The Anopheles gambiae TRPA1 (AgTRPA1) was found to be activated by heat and electrophilic compounds when reconstituted in artificial membranes after purification. The temperature activation as well as the binding of electrophilic ligands to AgTRPA1 resulted in the quenching of fluorescence suggesting that thermal and chemical activation brought about similar conformational changes of the protein and perhaps reflect the dynamic change in the conformation of residues involved in the gating process (Paper III). We also demonstrated that the N-terminal domain of both human and mosquito TRPA1 is not essential for thermal/chemical sensation (Paper III and Paper IV) as opposed to previous reports

Increased Permeability of the Aquaporin SoPIP2;1 by Mercury and Mutations in Loop A

Aquaporins (AQPs) also referred to as Major intrinsic proteins, regulate permeability of biological membranes for water and other uncharged small polar molecules. Plants encode more AQPs than other organisms and just one of the four AQP subfamilies in Arabidopsis thaliana, the water specific plasma membrane intrinsic proteins (PIPs), has 13 isoforms, the same number as the total AQPs encoded by the entire human genome. The PIPs are more conserved than other plant AQPs and here we demonstrate that a cysteine residue, in loop A of SoPIP2;1 from Spinacia oleracea, is forming disulfide bridges. This is in agreement with studies on maize PIPs, but in contrast we also show an increased permeability of mutants with a substitution at this position. In accordance with earlier findings, we confirm that mercury increases water permeability of both wild type and mutant proteins. We report on the slow kinetics and reversibility of the activation, and on quenching of intrinsic tryptophan fluorescence as a potential reporter of conformational changes associated with activation. Hence, previous studies in plants based on the assumption of mercury as a general AQP blocker have to be reevaluated, whereas mercury and fluorescence studies of isolated PIPs provide new means to follow structural changes dynamically

NMR resonance assignment and dynamics of profilin from Heimdallarchaeota

The origin of the eukaryotic cell is an unsettled scientific question. The Asgard superphylum has emerged as a compelling target for studying eukaryogenesis due to the previously unseen diversity of eukaryotic signature proteins. However, our knowledge about these proteins is still relegated to metagenomic data and very little is known about their structural properties. Additionally, it is still unclear if these proteins are functionally homologous to their eukaryotic counterparts. Here, we expressed, purified and structurally characterized profilin from Heimdallarchaeota in the Asgard superphylum. The structural analysis shows that while this profilin possesses similar secondary structural elements as eukaryotic profilin, it contains additional secondary structural elements that could be critical for its function and an indication of divergent evolution

The N-terminal Ankyrin Repeat domain is not required for electrophile and heat activation of the purified mosquito TRPA1 receptor

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that Ag- TRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli

A multisubstrate deoxyribonucleoside kinase from plants

Deoxyribonucleoside kinases catalyze the rate limiting step during the salvage of deoxyribonucleosides and convert them into the corresponding monophosphate compounds. We have identified and characterized a unique multisubstrate deoxyribonucleoside kinase from plants. The phylogenetic relationship and biochemical properties suggest that this deoxyribonucleoside kinase represents a living fossil resembling the progenitor of the modern animal deoxycytidine, deoxyguanosine and thymidine 2 kinases. The broad substrate specificity makes this enzyme an interesting candidate to be evaluated as a suicide gene in anti-cancer therapy

Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain.

We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease)

Heimdallarchaea encodes profilin with eukaryotic-like actin regulation and polyproline binding

It is now widely accepted that the first eukaryotic cell emerged from a merger of an archaeal host cell and an alphaproteobacterium. However, the exact sequence of events and the nature of the cellular biology of both partner cells is still contentious. Recently the structures of profilins from some members of the newly discovered Asgard superphylum were determined. In addition, it was found that these profilins inhibit eukaryotic rabbit actin polymerization and that this reaction is regulated by phospholipids. However, the interaction with polyproline repeats which are known to be crucial for the regulation of profilin:actin polymerization was found to be absent for these profilins and was thus suggested to have evolved later in the eukaryotic lineage. Here, we show that Heimdallarchaeota LC3, a candidate phylum within the Asgard superphylum, encodes a putative profilin (heimProfilin) that interacts with PIP2 and its binding is regulated by polyproline motifs, suggesting an origin predating the rise of the eukaryotes. More precisely, we determined the 3D-structure of Heimdallarchaeota LC3 profilin and show that this profilin is able to: i) inhibit eukaryotic actin polymerization in vitro; ii) bind to phospholipids; iii) bind to polyproline repeats from enabled/vasodilator-stimulated phosphoprotein; iv) inhibit actin from Heimdallarchaeota from polymerizing into filaments. Our results therefore provide hints of the existence of a complex cytoskeleton already in last eukaryotic common ancestor. Chi and coworkers characterise proteins of Heimdallarchaeeota LC3, a member of the Asgard super phylum, and specifically investigate heim-Profilin and heim-Actin, and their interactions with polyproline and phospholipids. They also determine the 3D-structure of Heimdallarchaeota LC3 profilin. Their results suggest that a complex cytoskeleton existed in the last eukaryotic common ancestor indicating an origin predating the rise of the eukaryotes

Phosphorylation of human aquaporin 2 (AQP2) allosterically controls its interaction with the lysosomal trafficking protein LIP5

The interaction between the renal water channel aquaporin-2 (AQP2) and the lysosomal trafficking regulator-interacting protein LIP5 targets AQP2 to multivesicular bodies and facilitates lysosomal degradation. This interaction is part of a process that controls AQP2 apical membrane abundance in a vasopressin-dependent manner, allowing for urine volume adjustment. Vasopressin regulates phosphorylation at four sites within the AQP2 C terminus (Ser256, Ser261, Ser264, and Thr269), of which Ser256 is crucial and sufficient for AQP2 translocation from storage vesicles to the apical membrane. However, whether AQP2 phosphorylation modulates AQP2-LIP5 complex affinity is unknown. Here we used far-Western blot analysis and microscale thermophoresis to show that the AQP2 binds LIP5 in a phosphorylation-dependent manner. We constructed five phospho-mimicking mutants (S256E, S261E, S264E, T269E, and S256E/T269E) and a C-terminal truncation mutant (ΔP242) that lacked all phosphorylation sites but retained a previously suggested LIP5-binding site. CD spectroscopy indicated that wild-type AQP2 and the phospho-mimicking mutants had similar overall structure but displayed differences in melting temperatures possibly arising from C-terminal conformational changes. Non-phosphorylated AQP2 bound LIP5 with the highest affinity, whereas AQP2-ΔP242 had 20-fold lower affinity as determined by microscale thermophoresis. AQP2-S256E, S261E, T269E, and S256E/T269E all had reduced affinity. This effect was most prominent for AQP2-S256E, which fits well with its role in apical membrane targeting. AQP2-S264E had affinity similar to non-phosphorylated AQP2, possibly indicating a role in exosome excretion. Our data suggest that AQP2 phosphorylation allosterically controls its interaction with LIP5, illustrating how altered affinities to interacting proteins form the basis for regulation of AQP2 trafficking by post-translational modifications