A widespread glutamine-sensing mechanism in the plant kingdom.

Glutamine is the primary metabolite of nitrogen assimilation from inorganic nitrogen sources in microorganisms and plants. The ability to monitor cellular nitrogen status is pivotal for maintaining metabolic homeostasis and sustaining growth. The present study identifies a glutamine-sensing mechanism common in the entire plant kingdom except Brassicaceae. The plastid-localized PII signaling protein controls, in a glutamine-dependent manner, the key enzyme of the ornithine synthesis pathway, N-acetyl-l-glutamate kinase (NAGK), that leads to arginine and polyamine formation. Crystal structures reveal that the plant-specific C-terminal extension of PII, which we term the Q loop, forms a low-affinity glutamine-binding site. Glutamine binding alters PII conformation, promoting interaction and activation of NAGK. The binding motif is highly conserved in plants except Brassicaceae. A functional Q loop restores glutamine sensing in a recombinant Arabidopsis thaliana PII protein, demonstrating the modular concept of the glutamine-sensing mechanism adopted by PII proteins during the evolution of plant chloroplasts.

Comparative Analysis of Single-Cell RNA Sequencing Methods with and without Sample Multiplexing.

Single-cell RNA sequencing (scRNA-seq) has emerged as a powerful technique for investigating biological heterogeneity at the single-cell level in human systems and model organisms. Recent advances in scRNA-seq have enabled the pooling of cells from multiple samples into single libraries, thereby increasing sample throughput while reducing technical batch effects, library preparation time, and the overall cost. However, a comparative analysis of scRNA-seq methods with and without sample multiplexing is lacking. In this study, we benchmarked methods from two representative platforms: Parse Biosciences (Parse; with sample multiplexing) and 10x Genomics (10x; without sample multiplexing). By using peripheral blood mononuclear cells (PBMCs) obtained from two healthy individuals, we demonstrate that demultiplexed scRNA-seq data obtained from Parse showed similar cell type frequencies compared to 10x data where samples were not multiplexed. Despite relatively lower cell capture affecting library preparation, Parse can detect rare cell types (e.g., plasmablasts and dendritic cells) which is likely due to its relatively higher sensitivity in gene detection. Moreover, a comparative analysis of transcript quantification between the two platforms revealed platform-specific distributions of gene length and GC content. These results offer guidance for researchers in designing high-throughput scRNA-seq studies.

The Pathogenesis of Systemic Sclerosis: The Origin of Fibrosis and Interlink with Vasculopathy and Autoimmunity.

Systemic sclerosis (SSc) is an autoimmune disease associated with increased mortality and poor morbidity, impairing the quality of life in patients. Whilst we know that SSc affects multiple organs via vasculopathy, inflammation, and fibrosis, its exact pathophysiology remains elusive. Microvascular injury and vasculopathy are the initial pathological features of the disease. Clinically, the vasculopathy in SSc is manifested as Raynaud's phenomenon (reversible vasospasm in reaction to the cold or emotional stress) and digital ulcers due to ischemic injury. There are several reports that medications for vasculopathy, such as bosentan and soluble guanylate cyclase (sGC) modulators, improve not only vasculopathy but also dermal fibrosis, suggesting that vasculopathy is important in SSc. Although vasculopathy is an important initial step of the pathogenesis for SSc, it is still unclear how vasculopathy is related to inflammation and fibrosis. In this review, we focused on the clinical evidence for vasculopathy, the major cellular players for the pathogenesis, including pericytes, adipocytes, endothelial cells (ECs), and myofibroblasts, and their signaling pathway to elucidate the relationship among vasculopathy, inflammation, and fibrosis in SSc.

From cyanobacteria to plants: conservation of PII functions during plastid evolution.

This article reviews the current state-of-the-art concerning the functions of the signal processing protein PII in cyanobacteria and plants, with a special focus on evolutionary aspects. We start out with a general introduction to PII proteins, their distribution, and their evolution. We also discuss PII-like proteins and domains, in particular, the similarity between ATP-phosphoribosyltransferase (ATP-PRT) and its PII-like domain and the complex between N-acetyl-L-glutamate kinase (NAGK) and its PII activator protein from oxygenic phototrophs. The structural basis of the function of PII as an ATP/ADP/2-oxoglutarate signal processor is described for Synechococcus elongatus PII. In both cyanobacteria and plants, a major target of PII regulation is NAGK, which catalyzes the committed step of arginine biosynthesis. The common principles of NAGK regulation by PII are outlined. Based on the observation that PII proteins from cyanobacteria and plants can functionally replace each other, the hypothesis that PII-dependent NAGK control was under selective pressure during the evolution of plastids of Chloroplastida and Rhodophyta is tested by bioinformatics approaches. It is noteworthy that two lineages of heterokont algae, diatoms and brown algae, also possess NAGK, albeit lacking PII; their NAGK however appears to have descended from an alphaproteobacterium and not from a cyanobacterium as in plants. We end this article by coming to the conclusion that during the evolution of plastids, PII lost its function in coordinating gene expression through the PipX-NtcA network but preserved its role in nitrogen (arginine) storage metabolism, and subsequently took over the fine-tuned regulation of carbon (fatty acid) storage metabolism, which is important in certain developmental stages of plants.

Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein.

P(II) proteins control key processes of nitrogen metabolism in bacteria, archaea, and plants in response to the central metabolites ATP, ADP, and 2-oxoglutarate (2-OG), signaling cellular energy and carbon and nitrogen abundance. This metabolic information is integrated by P(II) and transmitted to regulatory targets (key enzymes, transporters, and transcription factors), modulating their activity. In oxygenic phototrophs, the controlling enzyme of arginine synthesis, N-acetyl-glutamate kinase (NAGK), is a major P(II) target, whose activity responds to 2-OG via P(II). Here we show structures of the Synechococcus elongatus P(II) protein in complex with ATP, Mg(2+), and 2-OG, which clarify how 2-OG affects P(II)-NAGK interaction. P(II) trimers with all three sites fully occupied were obtained as well as structures with one or two 2-OG molecules per P(II) trimer. These structures identify the site of 2-OG located in the vicinity between the subunit clefts and the base of the T loop. The 2-OG is bound to a Mg(2+) ion, which is coordinated by three phosphates of ATP, and by ionic interactions with the highly conserved residues K58 and Q39 together with B- and T-loop backbone interactions. These interactions impose a unique T-loop conformation that affects the interactions with the P(II) target. Structures of P(II) trimers with one or two bound 2-OG molecules reveal the basis for anticooperative 2-OG binding and shed light on the intersubunit signaling mechanism by which P(II) senses effectors in a wide range of concentrations.

Toward Molecular Stratification and Precision Medicine in Systemic Sclerosis.

Systemic sclerosis (SSc), a complex multi-systemic disease characterized by immune dysregulation, vasculopathy and fibrosis, is associated with high mortality. Its pathogenesis is only partially understood. The heterogenous pathological processes that define SSc and its stages present a challenge to targeting appropriate treatment, with differing treatment outcomes of SSc patients despite similar initial clinical presentations. Timing of the appropriate treatments targeted at the underlying disease process is critical. For example, immunomodulatory treatments may be used for patients in a predominantly inflammatory phase, anti-fibrotic treatments for those in the fibrotic phase, or combination therapies for those in the fibro-inflammatory phase. In advancing personalized care through precision medicine, groups of patients with similar disease characteristics and shared pathological processes may be identified through molecular stratification. This would improve current clinical sub-setting systems and guide personalization of therapies. In this review, we will provide updates in SSc clinical and molecular stratification in relation to patient outcomes and treatment responses. Promises of molecular stratification through advances in high-dimensional tools, including omic-based stratification (transcriptomics, genomics, epigenomics, proteomics, cytomics, microbiomics) and machine learning will be discussed. Innovative and more granular stratification systems that integrate molecular characteristics to clinical phenotypes would potentially improve therapeutic approaches through personalized medicine and lead to better patient outcomes.

A novel signal transduction protein P(II) variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-P(II) complex formation.

P(II) signal transduction proteins are highly conserved in bacteria, archaea and plants and have key functions in coordination of central metabolism by integrating signals from the carbon, nitrogen and energy status of the cell. In the cyanobacterium Synechococcus elongatus PCC 7942, P(II) binds ATP and 2-oxoglutarate (2-OG) in a synergistic manner, with the ATP binding sites also accepting ADP. Depending on its effector molecule binding status, P(II) (from this cyanobacterium and other oxygenic phototrophs) complexes and regulates the arginine-controlled enzyme of the cyclic ornithine pathway, N-acetyl-l-glutamate kinase (NAGK), to control arginine biosynthesis. To gain deeper insights into the process of P(II) binding to NAGK, we searched for P(II) variants with altered binding characteristics and found P(II) variants I86N and I86T to be able to bind to an NAGK variant (R233A) that was previously shown to be unable to bind wild-type P(II) protein. Analysis of interactions between these P(II) variants and wild-type NAGK as well as with the NAGK R233A variant suggested that the P(II) I86N variant was a superactive NAGK binder. To reveal the structural basis of this property, we solved the crystal structure of the P(II) I86N variant at atomic resolution. The large T-loop, which prevails in most receptor interactions of P(II) proteins, is present in a tightly bended conformation that mimics the T-loop of S. elongatus P(II) after having latched onto NAGK. Moreover, both P(II) I86 variants display a specific defect in 2-OG binding, implying a role of residue I86 in 2-OG binding. We propose a two-step model for the mechanism of P(II)-NAGK complex formation: in an initiating step, a contact between R233 of NAGK and E85 of P(II) initiates the bending of the extended T-loop of P(II), followed by a second step, where a bended T-loop deeply inserts into the NAGK clefts to form the tight complex.