Quantitative assessment of the in-vitro binding kinetics of antisickling aromatic aldehydes with hemoglobin A: A universal HPLC-UV/Vis method to quantitate Schiff-base adduct formation.

Aromatic aldehydes act as allosteric effectors of hemoglobin (AEH), forming Schiff-base adducts with the protein to increase its oxygen (O2) affinity; a desirable property in sickle cell disease (SCD) treatment, as the high-O2 affinity hemoglobin (Hb) does not polymerize and subsequently prevents erythrocytes sickling. This study reports the development, validation, and application of a weak cation-exchange HPLC assay - quantifying the appearance of Hb-AEH adduct - as a "universal" method, allowing for the prioritization of AEH candidates through an understanding of their Hb binding affinity and kinetics. Concentration- and time-dependent Hb binding profiles of ten AEHs were determined with HPLC, followed by the appropriate non-linear modeling to characterize their steady-state binding affinity (KDss), and binding kinetics second-order association (kon) and first-order dissociation (koff) rate constants. Vanillin-derived AEHs exhibited enhanced binding affinity to Hb, primarily due to their faster kon. Across AEH, kon and koff values are strongly correlated (r = 0.993, n = 7), suggesting that modifications of the AEH scaffold enhanced their interactions with Hb as intended, but inadvertently increased their Hb-AEH adduct dissociation. To our knowledge, the present study is the first to provide valuable insight into Hb binding kinetics of antisickling aromatic aldehydes, and the assay will be a useful platform in screening/prioritizing drug candidates for SCD treatment.

Metabolic Reprogramming in Sickle Cell Diseases: Pathophysiology and Drug Discovery Opportunities.

Sickle cell disease (SCD) is a genetic disorder that affects millions of individuals worldwide. Chronic anemia, hemolysis, and vasculopathy are associated with SCD, and their role has been well characterized. These symptoms stem from hemoglobin (Hb) polymerization, which is the primary event in the molecular pathogenesis of SCD and contributes to erythrocyte or red blood cell (RBC) sickling, stiffness, and vaso-occlusion. The disease is caused by a mutation at the sixth position of the ß-globin gene, coding for sickle Hb (HbS) instead of normal adult Hb (HbA), which under hypoxic conditions polymerizes into rigid fibers to distort the shapes of the RBCs. Only a few therapies are available, with the universal effectiveness of recently approved therapies still being monitored. In this review, we first focus on how sickle RBCs have altered metabolism and then highlight how this understanding reveals potential targets involved in the pathogenesis of the disease, which can be leveraged to create novel therapeutics for SCD.

Mechanistic Analysis of the VirA Sensor Kinase in Agrobacterium tumefaciens Using Structural Models.

Agrobacterium tumefaciens pathogenesis of plants is initiated with signal reception and culminates with transforming the genomic DNA of its host. The histidine sensor kinase VirA receives and reacts to discrete signaling molecules for the full induction of the genes necessary for this process. Though many of the components of this process have been identified, the precise mechanism of how VirA coordinates the response to host signals, namely phenols and sugars, is unknown. Recent advances of molecular modeling have allowed us to test structure/function predictions and contextualize previous experiments with VirA. In particular, the deep mind software AlphaFold has generated a structural model for the entire protein, allowing us to construct a model that addresses the mechanism of VirA signal reception. Here, we deepen our analysis of the region of VirA that is critical for phenol reception, model and probe potential phenol-binding sites of VirA, and refine its mechanism to strengthen our understanding of A. tumefaciens signal perception.

Mapping Reaction-Diffusion Networks at the Plant Wound Site With Pathogens.

The rich collection of microbes colonizing the plant root making up the rhizosphere function as a multigenomic organ for nutrient distribution. The extent to which its dynamic mutualistic cellular order depends on morphogenic signaling, while likely, remains unknown. We have shown that reaction-diffusion chemical networks constructed with model plant and bacterial metabolites can mimic processes ranging from oxidative burst kinetics to traveling waves and extracellular stationary state reaction-diffusion networks for spatiotemporal ordering of the rhizosphere. Plant parasites and pathogens can be limited by host attachment require dynamic informational networks and continue to provide insight into what controls the rhizosphere. Here we take advantage of Agrobacterium tumefaciens, a plant pathogen with a gated receptor that requires simultaneous perception of two plant metabolites. Genetic manipulations have created receptors allowing each metabolite concentration to be correlated with pathogen behavior. The development of the florescent strains used here provide initial maps of the reaction-diffusion dynamics existing in the rhizosphere, revealing significant differences in the signaling landscape of host and non-host plants before and after wounding, specifically highlighting networks that may inform rhizosphere organization.

Redox-mediated quorum sensing in plants.

The rhizosphere, the narrow zone of soil around plant roots, is a complex network of interactions between plants, bacteria, and a variety of other organisms. The absolute dependence on host-derived signals, or xenognosins, to regulate critical developmental checkpoints for host commitment in the obligate parasitic plants provides a window into the rhizosphere's chemical dynamics. These sessile intruders use H2O2 in a process known as semagenesis to chemically modify the mature root surfaces of proximal host plants and generate p-benzoquinones (BQs). The resulting redox-active signaling network regulates the spatial and temporal commitments necessary for host attachment. Recent evidence from non-parasites, including Arabidopsis thaliana, establishes that reactive oxygen species (ROS) production regulates similar redox circuits related to root recognition, broadening xenognosins' role beyond the parasites. Here we compare responses to the xenognosin dimethoxybenzoquinone (DMBQ) between the parasitic plant Striga asiatica and the non-parasitic A. thaliana. Exposure to DMBQ simulates the proximity of a mature root surface, stimulating an increase in cytoplasmic Ca2+ concentration in both plants, but leads to remarkably different phenotypic responses in the parasite and non-parasite. In S. asiatica, DMBQ induces development of the host attachment organ, the haustorium, and decreases ROS production at the root tip, while in A. thaliana, ROS production increases and further growth of the root tip is arrested. Obstruction of Ca2+ channels and the addition of antioxidants both lead to a decrease in the DMBQ response in both parasitic and non-parasitic plants. These results are consistent with Ca2+ regulating the activity of NADPH oxidases, which in turn sustain the autocatalytic production of ROS via an external quinone/hydroquinone redox cycle. Mechanistically, this chemistry is similar to black and white photography with the emerging dynamic reaction-diffusion network laying the foundation for the precise temporal and spatial control underlying rhizosphere architecture.

A Rhizobium radiobacter Histidine Kinase Can Employ Both Boolean AND and OR Logic Gates to Initiate Pathogenesis.

The molecular logic gates that regulate gene circuits are necessarily intricate and highly regulated, particularly in the critical commitments necessary for pathogenesis. We now report simple AND and OR logic gates to be accessible within a single protein receptor. Pathogenesis by the bacterium Rhizobium radiobacter is mediated by a single histidine kinase, VirA, which processes multiple small molecule host signals (phenol and sugar). Mutagenesis analyses converged on a single signal integration node, and finer functional analyses revealed that a single residue could switch VirA from a functional AND logic gate to an OR gate where each of two signals activate independently. Host range preferences among natural strains of R. radiobacter correlate with these gate logic strategies. Although the precise mechanism for the signal integration node requires further analyses, long-range signal transmission through this histidine kinase can now be exploited for synthetic signaling circuits.

Role of the VirA histidine autokinase of Agrobacterium tumefaciens in the initial steps of pathogenesis.

Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.

Sequence of the yeast protein expression plasmid pEG(KT).

The plasmid pEG(KT) is a widely used plasmid for expressing high levels of GST fusion proteins in the yeast Saccharomyces cerevisiae. Unfortunately, a complete sequence file has been lacking, thus complicating efforts to design cloning projects or to modify the plasmid for other uses (e.g. exchanging selection markers, epitope tags or protease cleavage sites to remove the epitope tag). Here, the complete sequence of the pEG(KT) plasmid is reported, thus facilitating its use. Additionally, its use as a vector backbone for high-level expression of a TAP-tagged protein is shown.

Interaction of the endocytic scaffold protein Pan1 with the type I myosins contributes to the late stages of endocytosis.

The yeast endocytic scaffold Pan1 contains an uncharacterized proline-rich domain (PRD) at its carboxy (C)-terminus. We report that the pan1-20 temperature-sensitive allele has a disrupted PRD due to a frame-shift mutation in the open reading frame of the domain. To reveal redundantly masked functions of the PRD, synthetic genetic array screens with a pan1DeltaPRD strain found genetic interactions with alleles of ACT1, LAS17 and a deletion of SLA1. Through a yeast two-hybrid screen, the Src homology 3 domains of the type I myosins, Myo3 and Myo5, were identified as binding partners for the C-terminus of Pan1. In vitro and in vivo assays validated this interaction. The relative timing of recruitment of Pan1-green fluorescent protein (GFP) and Myo3/5-red fluorescent protein (RFP) at nascent endocytic sites was revealed by two-color real-time fluorescence microscopy; the type I myosins join Pan1 at cortical patches at a late stage of internalization, preceding the inward movement of Pan1 and its disassembly. In cells lacking the Pan1 PRD, we observed an increased lifetime of Myo5-GFP at the cortex. Finally, Pan1 PRD enhanced the actin polymerization activity of Myo5-Vrp1 complexes in vitro. We propose that Pan1 and the type I myosins interactions promote an actin activity important at a late stage in endocytic internalization.