Legume Alternative Oxidase Isoforms Show Differential Sensitivity to Pyruvate Activation

Alternative oxidase (AOX) is an important component of the plant respiratory pathway, enabling a route for electrons that bypasses the energy-conserving, ROS-producing complexes of the mitochondrial electron transport chain. Plants contain numerous isoforms of AOX, classified as either AOX1 or AOX2. AOX1 isoforms have received the most attention due to their importance in stress responses across a wide range of species. However, the propensity for at least one isoform of AOX2 to accumulate to very high levels in photosynthetic tissues of all legumes studied to date, suggests that this isoform has specialized roles, but we know little of its properties. Previous studies with sub-mitochondrial particles of soybean cotyledons and roots indicated that differential expression of GmAOX1, GmAOX2A, and GmAOX2D across tissues might confer different activation kinetics with pyruvate. We have investigated this using recombinantly expressed isoforms of soybean AOX in a previously described bacterial system (Selinski et al., 2016, Physiologia Plantarum 157, 264-279). Pyruvate activation kinetics were similar between the two GmAOX2 isoforms but differed substantially from those of GmAOX1, suggesting that selective expression of AOX1 and 2 could determine the level of AOX activity. However, this alone cannot completely explain the differences seen in sub-mitochondrial particles isolated from different legume tissues and possible reasons for this are discussed.

Hidden pathway for cytokine receptor activation: Structural insights into a marine sponge-derived lectin that activates the thrombopoietin receptor via recognition of the fucose moiety

N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism remains unstudied because no external agonist has been discovered. Here, we describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC). ThC-induced activation persists due to limited receptor internalization. Strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. We show that MPL is subject to sugar-mediated activation and that lectin-mediated activation kinetics differ from cytokine-mediated activation kinetics. Our data demonstrated the potential of lectins to provide deeper insight into human pathogenesis.

Epilepsy-causing KCNT1 variants increase KNa1.1 channel activity by disrupting the activation gate.

Gain-of-function pathogenic missense KCNT1 variants are associated with several developmental and epileptic encephalopathies (DEE). With few exceptions, patients are heterozygous and there is a paucity of mechanistic information about how pathogenic variants increase KNa1.1 channel activity and the behaviour of heterotetrameric channels comprising both wild-type (WT) and variant subunits. To better understand these, we selected a range of variants across the DEE spectrum, involving mutations in different protein domains and studied their functional properties. Whole-cell electrophysiology was used to characterise homomeric and heteromeric KNa1.1 channel assemblies carrying DEE-causing variants in the presence and absence of 10 mM intracellular sodium. Voltage-dependent activation of homomeric variant KNa1.1 assemblies were more hyperpolarised than WT KNa1.1 and, unlike WT KNa1.1, exhibited voltage-dependent activation in the absence of intracellular sodium. Heteromeric channels formed by co-expression of WT and variant KNa1.1 had activation kinetics intermediate of homomeric WT and variant KNa1.1 channels, with residual sodium-independent activity. In general, WT and variant KNa1.1 activation followed a single exponential, with time constants unaffected by voltage or sodium. Mutating the threonine in the KNa1.1 selectivity filter disrupted voltage-dependent activation, but sodium-dependence remained intact. Our findings suggest that KNa1.1 gating involves a sodium-dependent activation gate that modulates a voltage-dependent selectivity filter gate. Collectively, all DEE-associated KNa1.1 mutations lowered the energetic barrier for sodium-dependent activation, but some also had direct effects on selectivity filter gating. Destabilisation of the inactivated unliganded channel conformation can explain how DEE-causing amino acid substitutions in diverse regions of the channel structure all cause gain-of-function.

Both transient and sustained MPK3/6 activities positively control expression of NLR genes in PTI and ETI.

Arabidopsis thaliana Mitogen Activated Protein Kinases 3 and 6 (MPK3/6) are known to be activated transiently in PAMP-Triggered Immunity (PTI) and durably in Effector-Triggered Immunity (ETI). However the functional differences between these two kinds of activation kinetics and how they allow coordination of the two layers of plant immunity remain poorly understood. Here, by analysing suppressors of the phenotype caused by a constitutively active form of MPK3, we demonstrate that ETI-mediating nucleotide-binding domain leucine-rich repeat receptors (NLRs) and NLR signaling can act downstream of MPK3 activities. Moreover we provide evidence that both sustained and transient MPK3/6 activities positively control the expression of at least two NLR genes, AT3G04220 and AT4G1110. We further show that the ETI regulators NDR1 and EDS1 also contribute to the upregulations of these two NLRs not only in an ETI context but also in a PTI context. Remarkably, while in ETI, MPK3/6 activities are dependent on NDR1 and EDS1, they are not in PTI, suggesting that if the same actors are involved in the two layers of immunity, the way they are interconnected is different. Finally we demonstrate that expression of the NLR AT3G04220 is sufficient to induce expression of defense genes from the SA branch. Overall this study enlarges our knowledge of MPK3/6 functions during immunity and gives a new insight into the intrication of PTI and ETI.

Activation of electrospun carbon fibers: the effect of fiber diameter on CO2 and steam reaction kinetics

First, we present a fabrication process for electrospun carbon fiber mats with mean fiber diameters between 108 nm and 623 nm. The carbon fiber mats were produced by electrospinning of polyacrylonitrile (PAN) solutions and subsequent carbonization. The fiber mats feature small variations of their properties that are required for parameter studies. Second, we investigate the kinetics of steam and CO2 activation with three different activation temperatures and times. Both activation methods result in a surface area increase depending on activation temperature and time. Detailed analysis of the macroscopic properties burn-off, surface area, and conductivity reveals insights into the microscopic activation kinetics. The different fiber diameters of the carbon fiber mats enable the distinction of surface driven and bulk processes. Our results indicate, that CO2 activation kinetics are mass transport controlled, and that steam activation kinetics are reaction rate controlled. The turbostratic nature of PAN derived carbon and the distinct characteristics of the activation agents could explain the nonlinear behavior of the burn-off and surface area development.

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (NaV) and K (KV) channels. NaV channels activate much faster and allow the initial Na+ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in KV channels but an equally highly conserved threonine in NaV channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker KV channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of NaV channels comes from a stronger dielectric polarization by threonine (NaV channel) produced as the first gating charge enters the gating pore compared with isoleucine (KV channel).