Secreted Effectors Modulating Immune Responses to Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that chronically infects a third of humans. It can cause life-threatening encephalitis in immune-compromised individuals. Congenital infection also results in blindness and intellectual disabilities. In the intracellular milieu, parasites encounter various immunological effectors that have been shaped to limit parasite infection. Parasites not only have to suppress these anti-parasitic inflammatory responses but also ensure the host organism’s survival until their subsequent transmission. Recent advancements in T. gondii research have revealed a plethora of parasite-secreted proteins that suppress as well as activate immune responses. This mini-review will comprehensively examine each secreted immunomodulatory effector based on the location of their actions. The first section is focused on secreted effectors that localize to the parasitophorous vacuole membrane, the interface between the parasites and the host cytoplasm. Murine hosts are equipped with potent IFNγ-induced immune-related GTPases, and various parasite effectors subvert these to prevent parasite elimination. The second section examines several cytoplasmic and ER effectors, including a recently described function for matrix antigen 1 (MAG1) as a secreted effector. The third section covers the repertoire of nuclear effectors that hijack transcription factors and epigenetic repressors that alter gene expression. The last section focuses on the translocation of dense-granule effectors and effectors in the setting of T. gondii tissue cysts (the bradyzoite parasitophorous vacuole).

Programmable Sequences of Reflectin Proteins Provides Evidence for Their Evolution Processes, Tunable Intracellular Localization and Interaction with Cytoskeleton

Reflectins are membrane-bound proteins located in cephalopods iridocytes, with repeated canonical domains interspersed with cationic linkers. Scientists keep curious about their evolutionary processes, biochemical properties and intracellular functions. Here, by introducing reflectin A1, A2, B1 and C into HEK-293T cells, these proteins were found to phase out from the crowded intracellular milieu, with distinguished localization preferences. Inspired by their programmable block sequences, several truncated reflectin A1 (RfA1) peptides based on repetition of reflectin motifs were designed and transfected into cells. An obvious cyto-/nucleo-plasmic localization preference was once again observed. The dynamic performance of RfA1 derivatives and their analogic behavior between different reflectins suggest a conceivable evolutionary relationship among reflectin proteins. Additionally, a proteomic survey identified biochemical partners which contribute to the phase separation and intracellular localization of RfA1 and its truncations, as well as the close collaboration between RfA1 and the cytoskeleton systems. These findings indicate that liquid-liquid phase separation could be the fundamental mode for reflectins to achieve spatial organization, to cooperate with cytoskeleton during the regulation of reflective coloration. On the other hand, the dynamic behaviors of RfA1 derivatives strongly recommended themselves as programmable molecular tools.

A biochemically-realisable model of the self-manufacturing cell

As shown by Hofmeyr, the processes in the living cell can be divided into three classes of efficient causes that produce each other, so making the cell closed to efficient causation, the hallmark of an organism. They are the enzyme catalysts of covalent metabolic chemistry, the intracellular milieu that drives the supramolecular processes of chaperone-assisted folding and self-assembly of polypeptides and nucleic acids into functional catalysts and transporters, and the membrane transporters that maintain the intracellular milieu, in particular its electrolyte composition. Each class of efficient cause can be modelled as a relational diagram in the form of a mapping in graph-theoretic form, and a minimal model of a self-manufacturing system that is closed to efficient causation can be constructed from these three mappings using the formalism of relational biology. This Fabrication-Assembly or (F,A)-system serves as an alternative to Robert Rosen's replicative Metabolism-Repair or (M,R)-system, which has been notoriously problematic to realise in terms of real biochemical processes. A key feature of the model is the explicit incorporation of formal cause, which arrests the infinite regress that plagues all relational models of the cell. The (F,A)-system is extended into a detailed formal model of the self-manufacturing cell that has a clear biochemical realisation. This (F,A) cell model allows the interpretation and visualisation of concepts such as the metabolism and repair components of Rosen's (M,R)-system, John von Neumann's universal constructor, Howard Pattee's symbol-function split via the symbol-folding transformation, Marcello Barbieri's genotype-ribotype-phenotype ontology, and Tibor Gánti's chemoton. The (F,A) cell model also teaches us that, from the cell up to ecosystems, human organisations and societies, the internal context that allows members to function efficiently has agency, and should therefore be actively maintained from within by those very members.

Imaging the transmembrane and transendothelial sodium gradients in gliomas

Under normal conditions, high sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, and these manifest the cell membrane potential (Vm) as well as blood–brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic polyanionic agent to measure ΔNa+mem and ΔNa+end. In vitro 23Na-MRSI established that the 23Na signal is intensely shifted by the agent compared to other biological factors (e.g., pH and temperature). In vivo 23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together revealed weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas (vs. normal tissue). Compared to normal tissue, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, whereas RG2 tumors displayed elevated ∆Na+end suggesting altered BBB integrity. We anticipate that 23Na-MRSI will allow biomedical explorations of perturbed Na+ homeostasis in vivo.

Calculating metalation in cells reveals CobW acquires CoII for vitamin B12 biosynthesis while related proteins prefer ZnII

Protein metal-occupancy (metalation) in vivo has been elusive. To address this challenge, the available free energies of metals have recently been determined from the responses of metal sensors. Here, we use these free energy values to develop a metalation-calculator which accounts for inter-metal competition and changing metal-availabilities inside cells. We use the calculator to understand the function and mechanism of GTPase CobW, a predicted CoII-chaperone for vitamin B12. Upon binding nucleotide (GTP) and MgII, CobW assembles a high-affinity site that can obtain CoII or ZnII from the intracellular milieu. In idealised cells with sensors at the mid-points of their responses, competition within the cytosol enables CoII to outcompete ZnII for binding CobW. Thus, CoII is the cognate metal. However, after growth in different [CoII], CoII-occupancy ranges from 10 to 97% which matches CobW-dependent B12 synthesis. The calculator also reveals that related GTPases with comparable ZnII affinities to CobW, preferentially acquire ZnII due to their relatively weaker CoII affinities. The calculator is made available here for use with other proteins.

Emerging Roles of Long Noncoding RNAs in the Cytoplasmic Milieu

While the important functions of long noncoding RNAs (lncRNAs) in nuclear organization are well documented, their orchestrating and architectural roles in the cytoplasmic environment have long been underestimated. However, recently developed fractionation and proximity labelling approaches have shown that a considerable proportion of cellular lncRNAs is exported into the cytoplasm and associates nonrandomly with proteins in the cytosol and organelles. The functions of these lncRNAs range from the control of translation and mitochondrial metabolism to the anchoring of cellular components on the cytoskeleton and regulation of protein degradation at the proteasome. In the present review, we provide an overview of the functions of lncRNAs in cytoplasmic structures and machineries und discuss their emerging roles in the coordination of the dense intracellular milieu. It is becoming apparent that further research into the functions of these lncRNAs will lead to an improved understanding of the spatiotemporal organization of cytoplasmic processes during homeostasis and disease.

Imaging the Transmembrane and Transendothelial Sodium Gradients in Gliomas

ABSTRACTHigh sodium (Na+) in extracellular (Na+e) and blood (Na+b) compartments and low Na+ in intracellular milieu (Na+i) produce strong transmembrane (ΔNa+mem) and weak transendothelial (ΔNa+end) gradients respectively, which reflect cell membrane potential (Vm) and blood-brain barrier (BBB) integrity. We developed a sodium (23Na) magnetic resonance spectroscopic imaging (MRSI) method using an intravenously-administered paramagnetic contrast agent to measure ΔNa+mem and ΔNa+end. In vitro23Na-MRSI established that the 23Na signal is strongly shifted by the agent compared to biological factors. In vivo23Na-MRSI showed Na+i remained unshifted and Na+b was more shifted than Na+e, and these together created weakened ΔNa+mem and enhanced ΔNa+end in rat gliomas. Specifically, RG2 and U87 tumors maintained weakened ΔNa+mem (i.e., depolarized Vm) implying an aggressive state for proliferation, and RG2 tumors displayed elevated ΔNa+end suggesting altered BBB integrity. 23Na-MRSI will allow explorations of perturbed Na+ homeostasis in vivo for the tumor neurovascular unit.

Lysosome as a Central Hub for Rewiring PH Homeostasis in Tumors

Cancer cells generate large quantities of cytoplasmic protons as byproducts of aberrantly activated aerobic glycolysis and lactate fermentation. To avoid potentially detrimental acidification of the intracellular milieu, cancer cells activate multiple acid-removal pathways that promote cytosolic alkalization and extracellular acidification. Accumulating evidence suggests that in addition to the well-characterized ion pumps and exchangers in the plasma membrane, cancer cell lysosomes are also reprogrammed for this purpose. On the one hand, the increased expression and activity of the vacuolar-type H+−ATPase (V-ATPase) on the lysosomal limiting membrane combined with the larger volume of the lysosomal compartment increases the lysosomal proton storage capacity substantially. On the other hand, enhanced lysosome exocytosis enables the efficient release of lysosomal protons to the extracellular space. Together, these two steps dynamically drive proton flow from the cytosol to extracellular space. In this perspective, we provide mechanistic insight into how lysosomes contribute to the rewiring of pH homeostasis in cancer cells.