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Antiseptic mouthwash use is widespread due to its oral health benefits. However, its impact on systemic physiological processes, particularly nitric oxide (NO) bioavailability and muscle contractility, is not fully understood. We sought to determine the effects of cetylpyridinium (antibacterial) versus sodium chloride (control) mouthwashes on salivary and breath NO markers and muscle contractile function in healthy young adults. Thirty participants (n = 15/group) completed a randomized, parallel-arm, blinded trial, comparing the two mouthwashes before and after 7 d of treatment. NO bioavailability was inferred via measurement of salivary nitrate (NO 3 − ), nitrite (NO 2 − ), and cyclic guanyl monophosphate (cGMP) concentrations and breath NO level. Contractile function of the knee extensor muscles was determined via isokinetic dynamometry. No changes in salivary NO 3 − , NO 2 − , or cGMP or in breath NO were observed in response to either treatment. However, cetylpyridinium mouthwash reduced the percentage of NO 2 − in saliva (17 ± 10% vs. 25 ± 13%; p = 0.0036). Peak torque at velocities of 0–6.28 rad/s was unaffected by mouthwash use. Calculated maximal knee extensor velocity (Vmax) and power (Pmax) were therefore also unchanged. Cetylpyridinium mouthwash reduces the relative abundance of NO 2 − in the oral cavity but does not significantly diminish overall NO bioavailability or impair muscle contractile function in healthy young adults.

Robust associations between low plasma level of natriuretic peptides (NP) and increased risk of type 2 diabetes (T2D) have been recently reported in humans. Adipose tissue (AT) is a known target of NP. However it is unknown whether NP signalling in human AT relates to insulin sensitivity and modulates glucose metabolism. We here show in two European cohorts that the NP receptor guanylyl cyclase-A (GC-A) expression in subcutaneous AT was down-regulated as a function of obesity grade while adipose NP clearance receptor (NPRC) was up-regulated. Adipose GC-A mRNA level was down-regulated in prediabetes and T2D, and negatively correlated with HOMA-IR and fasting blood glucose. We show for the first time that NP promote glucose uptake in a dose-dependent manner. This effect is reduced in adipocytes of obese individuals. NP activate mammalian target of rapamycin complex 1/2 (mTORC1/2) and Akt signalling. These effects were totally abrogated by inhibition of cGMP-dependent protein kinase and mTORC1/2 by rapamycin. We further show that NP treatment favoured glucose oxidation and de novo lipogenesis independently of significant gene regulation. Collectively, our data support a role for NP in blood glucose control and insulin sensitivity by increasing glucose uptake in human adipocytes. This effect is partly blunted in obesity.

Because raising cAMP enhances 26S proteasome activity and the degradation of cell proteins, including the selective breakdown of misfolded proteins, we investigated whether agents that raise cGMP may also regulate protein degradation. Treating various cell lines with inhibitors of phosphodiesterase 5 or stimulators of soluble guanylyl cyclase rapidly enhanced multiple proteasome activities and cellular levels of ubiquitinated proteins by activating protein kinase G (PKG). PKG stimulated purified 26S proteasomes by phosphorylating a different 26S component than is modified by protein kinase A. In cells and cell extracts, raising cGMP also enhanced within minutes ubiquitin conjugation to cell proteins. Raising cGMP, like raising cAMP, stimulated the degradation of short-lived cell proteins, but unlike cAMP, also markedly increased proteasomal degradation of long-lived proteins (the bulk of cell proteins) without affecting lysosomal proteolysis. We also tested if raising cGMP, like cAMP, can promote the degradation of mutant proteins that cause neurodegenerative diseases. Treating zebrafish models of tauopathies or Huntington’s disease with a PDE5 inhibitor reduced the levels of the mutant huntingtin and tau proteins, cell death, and the resulting morphological abnormalities. Thus, PKG rapidly activates cytosolic proteasomes, protein ubiquitination, and overall protein degradation, and agents that raise cGMP may help combat the progression of neurodegenerative diseases.

Cyclic GMP (cGMP)-dependent protein kinase (protein kinase G [PKG]) is essential for microneme secretion, motility, invasion, and egress in apicomplexan parasites, However, the separate roles of two isoforms of the kinase that are expressed by some apicomplexans remain uncertain. Despite having identical regulatory and catalytic domains, PKG I is plasma membrane associated whereas PKG II is cytosolic in Toxoplasma gondii . To determine whether these isoforms are functionally distinct or redundant, we developed an auxin-inducible degron (AID) tagging system for conditional protein depletion in T. gondii . By combining AID regulation with genome editing strategies, we determined that PKG I is necessary and fully sufficient for PKG-dependent cellular processes. Conversely, PKG II is functionally insufficient and dispensable in the presence of PKG I . The difference in functionality mapped to the first 15 residues of PKG I , containing a myristoylated Gly residue at position 2 that is critical for membrane association and PKG function. Collectively, we have identified a novel requirement for cGMP signaling at the plasma membrane and developed a new system for examining essential proteins in T. gondii . IMPORTANCE Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii . We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii . Toxoplasma gondii is an obligate intracellular apicomplexan parasite and important clinical and veterinary pathogen that causes toxoplasmosis. Since apicomplexans can only propagate within host cells, efficient invasion is critically important for their life cycles. Previous studies using chemical genetics demonstrated that cyclic GMP signaling through protein kinase G (PKG)-controlled invasion by apicomplexan parasites. However, these studies did not resolve functional differences between two compartmentalized isoforms of the kinase. Here we developed a conditional protein regulation tool to interrogate PKG isoforms in T. gondii . We found that the cytosolic PKG isoform was largely insufficient and dispensable. In contrast, the plasma membrane-associated isoform was necessary and fully sufficient for PKG function. Our studies identify the plasma membrane as a key location for PKG activity and provide a broadly applicable system for examining essential proteins in T. gondii .

Biofilm formation protects bacteria from stresses including antibiotics and host immune responses. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae , but the underlying mechanisms remain unclear. Here, we show that EIIA Glc , a component of the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), regulates the intracellular concentration of the cyclic dinucleotide c-di-GMP, and thus biofilm formation. The availability of preferred sugars such as glucose affects EIIA Glc phosphorylation state, which in turn modulates the interaction of EIIA Glc with a c-di-GMP phosphodiesterase (hereafter referred to as PdeS). In a Drosophila model of V. cholerae infection, sugars in the host diet regulate gut colonization in a manner dependent on the PdeS-EIIA Glc interaction. Our results shed light into the mechanisms by which some nutrients regulate biofilm formation and host colonization. Carbon sources can modulate biofilm formation and host colonization in Vibrio cholerae . Here, Heo et al. show that this process is mediated by a component of the PEP:carbohydrate phosphotransferase system (PTS), which regulates c-di-GMP hydrolysis by interacting with a c-di-GMP phosphodiesterase.

Metabolic reprogramming to fulfill the biosynthetic and bioenergetic demands of cancer cells has aroused great interest in recent years. However, metabolic reprogramming for cancer metastasis has not been well elucidated. Here, we screened a subpopulation of breast cancer cells with highly metastatic capacity to the lung in mice and investigated the metabolic alternations by analyzing the metabolome and the transcriptome, which were confirmed in breast cancer cells, mouse models, and patients’ tissues. The effects and the mechanisms of nucleotide de novo synthesis in cancer metastasis were further evaluated in vitro and in vivo. In our study, we report an increased nucleotide de novo synthesis as a key metabolic hallmark in metastatic breast cancer cells and revealed that enforced nucleotide de novo synthesis was enough to drive the metastasis of breast cancer cells. An increased key metabolite of de novo synthesis, guanosine-5'-triphosphate (GTP), is able to generate more cyclic guanosine monophosphate (cGMP) to activate cGMP-dependent protein kinases PKG and downstream MAPK pathway, resulting in the increased tumor cell stemness and metastasis. Blocking de novo synthesis by silencing phosphoribosylpyrophosphate synthetase 2 (PRPS2) can effectively decrease the stemness of breast cancer cells and reduce the lung metastasis. More interestingly, in breast cancer patients, the level of plasma uric acid (UA), a downstream metabolite of purine, is tightly correlated with patient’s survival. Our study uncovered that increased de novo synthesis is a metabolic hallmark of metastatic breast cancer cells and its metabolites can regulate the signaling pathway to promote the stemness and metastasis of breast cancer.

BLUF photoreceptor: light-activated scissors BLUF is a photoreceptor protein domain that uses an FAD chromophore to sense blue light. Although X-ray crystal structures of single-domain BLUF proteins have been determined, there have not been any reports of a structure of a BLUF protein that also contained a functional 'output' domain. For this reason, the mechanism(s) of light activation for this class of photoreceptors has remained enigmatic. Here, Thomas Barends and colleagues report the first biochemical, structural, and mechanistic characterization of a full-length, active photoreceptor. The protein is from the bacterium Klebsiella pneumoniae , and it contains the BLUF sensor domain and a phosphodiesterase output domain that hydrolyses cyclic dimeric GMP. The structures of this protein co-complexed with its substrate and metal ions provide a detailed understanding of how light absorbed by the BLUF domain leads to activation of the phosphodiesterase output domain. Although structures of single-domain BLUF proteins—a photoreceptor protein domain that senses blue light—have been determined, there have been no reports of the structure of a BLUF protein containing a functional output domain; for this reason, the mechanism of light activation has remained enigmatic. The first biochemical, structural and mechanistic characterization of a full-length, active photoreceptor containing a BLUF sensor domain and a phosphodiesterase EAL output domain is now reported. The ability to respond to light is crucial for most organisms. BLUF is a recently identified photoreceptor protein domain that senses blue light using a FAD chromophore 1 . BLUF domains are present in various proteins from the Bacteria, Euglenozoa and Fungi. Although structures of single-domain BLUF proteins have been determined 2 , 3 , 4 , none are available for a BLUF protein containing a functional output domain; the mechanism of light activation in this new class of photoreceptors has thus remained poorly understood. Here we report the biochemical, structural and mechanistic characterization of a full-length, active photoreceptor, BlrP1 (also known as KPN_01598), from Klebsiella pneumoniae 5 . BlrP1 consists of a BLUF sensor domain and a phosphodiesterase EAL output domain which hydrolyses cyclic dimeric GMP (c-di-GMP). This ubiquitous second messenger controls motility, biofilm formation, virulence and antibiotic resistance in the Bacteria 6 , 7 , 8 , 9 . Crystal structures of BlrP1 complexed with its substrate and metal ions involved in catalysis or in enzyme inhibition provide a detailed understanding of the mechanism of the EAL-domain c-di-GMP phosphodiesterases. These structures also sketch out a path of light activation of the phosphodiesterase output activity. Photon absorption by the BLUF domain of one subunit of the antiparallel BlrP1 homodimer activates the EAL domain of the second subunit through allosteric communication transmitted through conserved domain–domain interfaces.

The second messenger cyclic dimeric GMP (c-di-GMP) regulates surface attachment and biofilm formation by many bacteria. For Pseudomonas fluorescens Pf0-1, c-di-GMP impacts the secretion and localization of the adhesin LapA, which is absolutely required for stable surface attachment and biofilm formation by this bacterium. In this study we characterize LapD, a unique c-di-GMP effector protein that controls biofilm formation by communicating intracellular c-di-GMP levels to the membrane-localized attachment machinery via its periplasmic domain. LapD contains degenerate and enzymatically inactive diguanylate cyclase and c-di-GMP phosphodiesterase (EAL) domains and binds to c-di-GMP through a degenerate EAL domain. We present evidence that LapD utilizes an inside-out signaling mechanism: binding c-di-GMP in the cytoplasm and communicating this signal to the periplasm via its periplasmic domain. Furthermore, we show that LapD serves as the c-di-GMP receptor connecting environmental modulation of intracellular c-di-GMP levels by inorganic phosphate to regulation of LapA localization and thus surface commitment by P. fluorescens.

It is now clear that cell-cell communication, often referred to as quorum sensing (QS), is the norm in the prokaryotic kingdom and this community-wide genetic regulatory mechanism has been adopted for regulation of many important biological functions. Since the 1980s, several types of QS signals have been identified, which are associated commonly with different types of QS mechanisms. Among them, the diffusible signal factor (DSF)-dependent QS system, originally discovered from bacterial pathogen Xanthomonas campestris pv. campestris, is a relatively new regulatory mechanism. The rapid research progress over the last few years has identified the chemical structure of the QS signal DSF, established the DSF regulon, and unveiled the general signaling pathways and mechanisms. Particular noteworthy are that DSF biosynthesis is modulated by a novel posttranslational autoinduction mechanism involving protein-protein interaction between the DSF synthase RpfF and the sensor RpfC, and that QS signal sensing is coupled to intracellular regulatory networks through a second messenger cyclic-di-GMP and a global regulator Clp. Genomic and genetic analyses show that the DSF QS-signaling pathway regulates diverse biological functions including virulence, biofilm dispersal, and ecological competence. Moreover, evidence is emerging that the DSF QS system is conserved in a range of plant and human bacterial pathogens.

Hydrogen sulfide (H₂S) is a unique gasotransmitter, with regulatory roles in the cardiovascular, nervous, and immune systems. Some of the vascular actions of H₂S (stimulation of angiogenesis, relaxation of vascular smooth muscle) resemble those of nitric oxide (NO). Although it was generally assumed that H₂S and NO exert their effects via separate pathways, the results of the current study show that H₂S and NO are mutually required to elicit angiogenesis and vasodilatation. Exposure of endothelial cells to H₂S increases intracellular cyclic guanosine 5'-monophosphate (cGMP) in a NO-dependent manner, and activated protein kinase G (PKG) and its downstream effector, the vasodilator-stimulated phosphoprotein (VASP). Inhibition of endothelial isoform of NO synthase (eNOS) or PKG-I abolishes the H₂S-stimulated angiogenic response, and attenuated H₂S-stimulated vasorelaxation, demonstrating the requirement of NO in vascular H₂S signaling. Conversely, silencing of the H₂S-producing enzyme cystathionme-γ-lyase abolishes NO-stimulated cGMP accumulation and angiogenesis and attenuates the acetylcholine-induced vasorelaxation, indicating a partial requirement of H₂S in the vascular activity of NO. The actions of H₂S and NO converge at cGMP; though H₂S does not directly activate soluble guanylyl cyclase, it maintains a tonic inhibitory effect on PDE5, thereby delaying the degradation of cGMP. H₂S also activates PI3K/Akt, and increases eNOS phosphorylation at its activating site S1177. The cooperative action of the two gasotransmitters on increasing and maintaining intracellular cGMP is essential for PKG activation and angiogenesis and vasorelaxation. H₂S-induced wound healing and microvessel growth in matrigel plugs is suppressed by pharmacological inhibition or genetic ablation of eNOS. Thus, NO and H₂S are mutually required for the physiological control of vascular function.