Heterotrimeric G Proteins in Plants: Canonical and Atypical Gα Subunits

Heterotrimeric GTP-binding proteins (G proteins), consisting of Gα, Gβ and Gγ subunits, transduce signals from a diverse range of extracellular stimuli, resulting in the regulation of numerous cellular and physiological functions in Eukaryotes. According to the classic G protein paradigm established in animal models, the bound guanine nucleotide on a Gα subunit, either guanosine diphosphate (GDP) or guanosine triphosphate (GTP) determines the inactive or active mode, respectively. In plants, there are two types of Gα subunits: canonical Gα subunits structurally similar to their animal counterparts and unconventional extra-large Gα subunits (XLGs) containing a C-terminal domain homologous to the canonical Gα along with an extended N-terminal domain. Both Gα and XLG subunits interact with Gβγ dimers and regulator of G protein signalling (RGS) protein. Plant G proteins are implicated directly or indirectly in developmental processes, stress responses, and innate immunity. It is established that despite the substantial overall similarity between plant and animal Gα subunits, they convey signalling differently including the mechanism by which they are activated. This review emphasizes the unique characteristics of plant Gα subunits and speculates on their unique signalling mechanisms.

Metabolites Profiling of Melanoma Interstitial Fluids Reveals Uridine Diphosphate as Potent Immune Modulator Capable of Limiting Tumor Growth

Tumor interstitial fluid (TIF) surrounds and perfuses tumors and collects ions, metabolites, proteins, and extracellular vesicles secreted by tumor and stromal cells. Specific metabolites, accumulated within the TIF, could induce metabolic alterations of immune cells and shape the tumor microenvironment. We deployed a metabolomic approach to analyze the composition of melanoma TIF and compared it to the plasma of C57BL6 mice, engrafted or not with B16-melanoma cells. Among the classes of metabolites analyzed, monophosphate and diphosphate nucleotides resulted enriched in TIF compared to plasma samples. The analysis of the effects exerted by guanosine diphosphate (GDP) and uridine diphosphate (UDP) on immune response revealed that GDP and UDP increased the percentage of CD4+CD25+FoxP3– and, on isolated CD4+ T-cells, induced the phosphorylation of ERK, STAT1, and STAT3; increased the activity of NF-κB subunits p65, p50, RelB, and p52; increased the expression of Th1/Th17 markers including IFNγ, IL17, T-bet, and RORγt; and reduced the expression of IL13, a Th2 marker. Finally, we observed that local administrations of UDP in B16-engrafted C57BL6 mice reduced tumor growth and necrotic areas. In addition, UDP-treated tumors showed a higher presence of MHCIIhi tumor-associated macrophage (TAM) and of CD3+CD8+ and CD3+CD4+ tumor-infiltrating T-lymphocytes (TILs), both markers of anti-tumor immune response. Consistent with this, intra-tumoral gene expression analysis revealed in UDP-treated tumors an increase in the expression of genes functionally linked to anti-tumor immune response. Our analysis revealed an important metabolite acting as mediator of immune response, which could potentially represent an additional tool to be used as an adjuvant in cancer immunotherapy.

F-box protein MdAMR1L1 regulates ascorbate biosynthesis in apple by modulating GDP-mannose pyrophosphorylase

Ascorbate (Asc) is an important antioxidant in plants and humans that plays key roles in various physiological processes. Understanding the regulation of Asc content in fruit plants is important for improving plant resiliency and optimizing Asc in food. Here, we found that both the transcript level and protein abundance of Asc Mannose pathway Regulator 1 Like 1 (MdAMR1L1) was negatively associated with Asc levels during the development of apple (Malus × domestica) fruit. The overexpression or silencing of MdAMR1L1 in apple indicated that MdAMR1L1 negatively regulated Asc levels. However, in the leaves of MdAMR1L1-overexpressing apple lines, the transcript levels of the Asc synthesis gene Guanosine diphosphate-mannose pyrophosphorylase MdGMP1 were increased, while its protein levels and enzyme activity were reduced. This occurred because the MdAMR1L1 protein interacted with MdGMP1 and promoted its degradation via the ubiquitination pathway to inhibit Asc synthesis at the post-translational level. MdERF98, an apple ethylene response factor, whose transcription was modulated by Asc level, is directly bound to the promoter of MdGMP1 to promote the transcription of MdGMP1. These findings provide insights into the regulatory mechanism of Asc biosynthesis in apples and revealed potential opportunities to improve fruit Asc levels.

On the asymmetric partitioning of nucleocytoplasmic transport – recent insights and open questions

ABSTRACT Macromolecular cargoes are asymmetrically partitioned in the nucleus or cytoplasm by nucleocytoplasmic transport (NCT). At the center of this activity lies the nuclear pore complex (NPC), through which soluble factors circulate to orchestrate NCT. These include cargo-carrying importin and exportin receptors from the β-karyopherin (Kapβ) family and the small GTPase Ran, which switches between guanosine triphosphate (GTP)- and guanosine diphosphate (GDP)-bound forms to regulate cargo delivery and compartmentalization. Ongoing efforts have shed considerable light on how these soluble factors traverse the NPC permeability barrier to sustain NCT. However, this does not explain how importins and exportins are partitioned in the cytoplasm and nucleus, respectively, nor how a steep RanGTP–RanGDP gradient is maintained across the nuclear envelope. In this Review, we peel away the multiple layers of control that regulate NCT and juxtapose unresolved features against known aspects of NPC function. Finally, we discuss how NPCs might function synergistically with Kapβs, cargoes and Ran to establish the asymmetry of NCT.

Unveiling the “invisible” druggable conformations of GDP-bound inactive Ras

The prevalent view on whether Ras is druggable has gradually changed in the recent decade with the discovery of effective inhibitors binding to cryptic sites unseen in the native structures. Despite the promising advances, therapeutics development toward higher potency and specificity is challenged by the elusive nature of these binding pockets. Here we derive a conformational ensemble of guanosine diphosphate (GDP)-bound inactive Ras by integrating spin relaxation-validated atomistic simulation with NMR chemical shifts and residual dipolar couplings, which provides a quantitative delineation of the intrinsic dynamics up to the microsecond timescale. The experimentally informed ensemble unequivocally demonstrates the preformation of both surface-exposed and buried cryptic sites in Ras•GDP, advocating design of inhibition by targeting the transient druggable conformers that are invisible to conventional experimental methods. The viability of the ensemble-based rational design has been established by retrospective testing of the ability of the Ras•GDP ensemble to identify known ligands from decoys in virtual screening.

Characterization of GMPP from Dendrobium huoshanense yielding GDP-D-mannose

Dendrobium huoshanense has been used for centuries in China and its polysaccharides are the main active components in treating loss of body fluids resulting from fever and asthenic symptoms. However, the biosynthetic pathway of polysaccharides in D. huoshanense remains to be elucidated. In this study, we obtained a guanosine diphosphate (GDP)-mannose pyrophosphorylase (DhGMPP) from D. huoshanense and characterized its function to catalyze the conversion of α-D-mannose-phosphate to GDP-D-mannose involved in the production of polysaccharides. DhGMPP, with the open reading frame of 1,245 bp, was isolated from RNA-Seq data of D. huoshanense. Phylogenetic analysis as well as sequence characterization suggested its involvement in the biosynthesis of GDP-D-mannose. In vitro enzyme assay demonstrated that GMPP encoded a pyrophosphorylase that converted α-D-mannose-phosphate and GTP into GDP-D-mannose. Identification of DhGMPP could provide more insights into the mechanism concerning polysaccharide biosynthesis in D. huoshanense and be utilized for enhancing polysaccharide accumulation through metabolic engineering.