https://www.cell.com/neuron/pdf/S0896-6273(14)01088-5.pdf

The Roles of PINK1, Parkin and Mitochondrial Fidelity in Parkinson's Disease

The IL-6/JAK/STAT3 pathway is aberrantly hyperactivated in many types of cancer, and such hyperactivation is generally associated with a poor clinical prognosis In the tumour microenvironment, IL-6/JAK/STAT3 signalling acts to drive the proliferation, survival, invasiveness, and metastasis of tumour cells, while strongly suppressing the antitumour immune response Thus, treatments that target the IL-6/JAK/STAT3 pathway in patients with cancer are poised to provide therapeutic benefit by directly inhibiting tumour cell growth and by stimulating antitumour immunity Agents targeting IL-6, the IL-6 receptor, or JAKs have already received FDA approval for the treatment of inflammatory conditions or myeloproliferative neoplasms and for the management of certain adverse effects of chimeric antigen receptor T cells, and are being further evaluated in patients with haematopoietic malignancies and in those with solid tumours Novel inhibitors of the IL-6/JAK/STAT3 pathway, including STAT3-selective inhibitors, are currently in development Herein, we review the role of IL-6/JAK/STAT3 signalling in the tumour microenvironment and the status of preclinical and clinical investigations of agents targeting this pathway We also discuss the potential of combining IL-6/JAK/STAT3 inhibitors with currently approved therapeutic agents directed against immune-checkpoint inhibitors

Targeting the IL-6/JAK/STAT3 signalling axis in cancer.

SUMMARY Twenty-five years have passed since the discovery of cyclic dimeric (3′→5′) GMP (cyclic di-GMP or c-di-GMP). From the relative obscurity of an allosteric activator of a bacterial cellulose synthase, c-di-GMP has emerged as one of the most common and important bacterial second messengers. Cyclic di-GMP has been shown to regulate biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes. Most c-di-GMP-dependent signaling pathways control the ability of bacteria to interact with abiotic surfaces or with other bacterial and eukaryotic cells. Cyclic di-GMP plays key roles in lifestyle changes of many bacteria, including transition from the motile to the sessile state, which aids in the establishment of multicellular biofilm communities, and from the virulent state in acute infections to the less virulent but more resilient state characteristic of chronic infectious diseases. From a practical standpoint, modulating c-di-GMP signaling pathways in bacteria could represent a new way of controlling formation and dispersal of biofilms in medical and industrial settings. Cyclic di-GMP participates in interkingdom signaling. It is recognized by mammalian immune systems as a uniquely bacterial molecule and therefore is considered a promising vaccine adjuvant. The purpose of this review is not to overview the whole body of data in the burgeoning field of c-di-GMP-dependent signaling. Instead, we provide a historic perspective on the development of the field, emphasize common trends, and illustrate them with the best available examples. We also identify unresolved questions and highlight new directions in c-di-GMP research that will give us a deeper understanding of this truly universal bacterial second messenger.

Cyclic di-GMP: the First 25 Years of a Universal Bacterial Second Messenger

On the stage of bacterial signal transduction and regulation, bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP) has long played the part of Sleeping Beauty. c-di-GMP was first described in 1987, but only recently was it recognized that the enzymes that 'make and break' it are not only ubiquitous in the bacterial world, but are found in many species in huge numbers. As a key player in the decision between the motile planktonic and sedentary biofilm-associated bacterial 'lifestyles', c-di-GMP binds to an unprecedented range of effector components and controls diverse targets, including transcription, the activities of enzymes and larger cellular structures. This Review focuses on emerging principles of c-di-GMP signalling using selected systems in different bacteria as examples.

Principles of c-di-GMP signalling in bacteria.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

Increases in S-nitrosylation and inactivation of the neuroprotective ubiquitin E3 ligase, parkin, in the brains of patients with Parkinson's disease are thought to be pathogenic and suggest a possible mechanism linking parkin to sporadic Parkinson's disease. Here we demonstrate that physiologic modification of parkin by hydrogen sulfide, termed sulfhydration, enhances its catalytic activity. Sulfhydration sites are identified by mass spectrometry analysis and are investigated by site-directed mutagenesis. Parkin sulfhydration is markedly depleted in the brains of patients with Parkinson's disease, suggesting that this loss may be pathologic. This implies that hydrogen sulfide donors may be therapeutic.

/pdf/sulfhydration-mediates-neuroprotective-actions-of-parkin-4iritwtqo3.pdf

Sulfhydration mediates neuroprotective actions of parkin

Erratum: Inositol polyphosphate multikinase is a coactivator of p53-mediated transcription and cell death (Science Signaling (2013) 6: 273 (er1) Doi: 10.1126/scisignal.6273er1)

Cyclic di-GMP was first discovered as a regulator of cellulose synthesis in Glucoacetobacter xylinus and is emerging as a major bacterial second messenger (14, 27, 28, 37). The intracellular concentration of cyclic di-GMP is controlled by the GGDEF domain proteins with diguanylate cyclase (DGC) activity and the EAL domain proteins with cyclic di-GMP-specific phosphodiesterase activity (27). GGDEF domains catalyze the condensation of two molecules of GTP to generate cyclic di-GMP, while the EAL domains catalyze the hydrolysis of cyclic di-GMP to generate the dinucleotide 5′-pGpG (Fig. ​(Fig.1).1). A family of HD-GYP domain proteins is also able to hydrolyze cyclic di-GMP to produce GMP (30), but the overwhelmingly large number of genes encoding the EAL domains in bacterial genomes suggests that they are the major phosphodiesterases responsible for maintaining the cellular cyclic di-GMP concentration.



FIG. 1.

Phosphodiesterase A (PDE-A) catalyzes hydrolysis of cyclic-di-GMP to generate the linear diguanylic acid 5′-pGpG.



Accumulating evidence suggests that EAL domain-containing proteins, including a large number of proteins that contain both the EAL and GGDEF domains, regulate a variety of cellular functions and phenotypes associated with bacterial infection. The regulation of virulence gene transcription, biofilm formation, motility, and adhesion has been reported in various pathogenic bacteria. Several proteins in the human pathogen Vibrio cholerae, including VieA and CdgC, have been implicated in biofilm formation, motility, and virulence factor production (15, 23, 40). An EAL domain protein was found to control lateral flagellar-gene expression and swarming behavior in Vibrio parahaemolyticus (17). In Salmonella enterica, the disruption of the EAL domain protein CdgR weakens bacterial resistance to hydrogen peroxide and accelerates bacterial killing by macrophages (12). In the opportunistic pathogen Pseudomonas aeruginosa, the EAL domain-containing protein FimX controls twitching motility and biofilm formation (13, 16) and the BifA protein controls biofilm formation and swarming (19). A systematic analysis of the GGDEF and EAL domain proteins in P. aeruginosa identified several other EAL domain proteins as being involved in virulence expression and biofilm formation (16, 20). Therefore, although the EAL domain proteins are not essential for the in vitro viability of pathogenic bacteria, they may be critical for the in vivo survival of the pathogens in host organisms, considering their roles in virulence expression and biofilm formation. This makes them potential targets for developing antibacterial agents that aim to neutralize virulence functions.

The genomes of the bacteria that contain cyclic di-GMP signaling networks generally encode multiple EAL domain proteins. For example, the genomes of P. aeruginosa PAO-1 and V. cholerae contain 21 and 32 open reading frames, respectively, for EAL domain proteins. In previous biochemical studies of EAL domain proteins, it was shown that Mg2+, or Mn2+, is required for the enzymes to hydrolyze cyclic di-GMP (31, 36, 38). It was also found that Zn2+ and Ca2+ can strongly inhibit the enzymatic activity, presumably by dislodging the Mg2+ ion. The Glu in the EAL (or EXL) signature motif seems to be essential for the enzymatic activity, because the E→A mutations in two EAL domain proteins abolished their phosphodiesterase activities (6, 38). Additionally, Schmidt and coworkers suggested that other conserved motifs, including a DDFGTG motif, may be essential for the catalytic activity (31). The phosphodiesterase activity of the EAL domain of CC3396 from Caulobacter crescentus can be stimulated by the binding of GTP to the adjacent enzymatically inactive GGDEF domain (6). A similar observation was reported for the protein FimX, involved in the regulation of twitching motion in P. aeruginosa (16). The utilization of the GGDEF domain could be a major strategy for regulating the catalytic activity of EAL domains, considering the large number of proteins that contain the GGDEF-EAL didomain. Meanwhile, the widespread occurrence of GGDEF-EAL domains also raises the possibility that some EAL domains may function as regulatory rather than catalytic domains. However, due to limited information about the catalytic mechanism of EAL domains, such a distinction has remained speculative.

RocR was identified as a response regulator in the RocSAR (or SadARS) two-component signaling system in P. aeruginosa (18, 21, 29). RocSAR consists of the histidine kinase RocS1 and two response regulators, RocA1 and RocR. The RocSAR system controls bacterial biofilm formation and virulence gene expression by regulating the transcription of various genes, including the cup fimbrial-gene clusters and type III secretion system genes (18, 20, 21). Deduced from the protein sequence, RocR contains an N-terminal CheY-like phosphoryl receiver domain and a C-terminal EAL domain. It was postulated that RocR negatively regulates the expression of cup genes by antagonizing the activity of RocA1, which is a typical response regulator with a DNA-binding domain (21). The detailed molecular mechanism for this antagonism is not known at present, though it has been speculated that the EAL domain may function as a regulatory domain lacking phosphodiesterase activity. Here, we present biochemical data to demonstrate that the EAL domain of RocR is catalytically active, with cyclic di-GMP-specific phosphodiesterase activity. Using RocR as a model system, we carried out systematic mutagenesis in the EAL domain to probe the roles of 14 conserved polar residues in catalysis. Based on the biochemical data and aided by the crystal structure of a homologous EAL domain protein, we assigned functions to the conserved residues and proposed a general base-catalyzed mechanism with the assistance of the Mg2+ ion. In the context of the proposed catalytic mechanism, we rationalize the inactivity of some characterized EAL domains and discuss the possibility of predicting the phosphodiesterase activities of EAL domains based on protein sequences.

Catalytic Mechanism of Cyclic Di-GMP-Specific Phosphodiesterase: a Study of the EAL Domain-Containing RocR from Pseudomonas aeruginosa

YybT is a Signaling Protein that Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (/)8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (Km) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in Km correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg 2 ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

https://www.genscript.com/site2/document/10207_20090716000434.PDF

Feng Rao

Papers

Sulfhydration mediates neuroprotective actions of parkin

Erratum: Inositol polyphosphate multikinase is a coactivator of p53-mediated transcription and cell death (Science Signaling (2013) 6: 273 (er1) Doi: 10.1126/scisignal.6273er1)

Catalytic Mechanism of Cyclic Di-GMP-Specific Phosphodiesterase: a Study of the EAL Domain-Containing RocR from Pseudomonas aeruginosa

YybT is a Signaling Protein that Contains a Cyclic Dinucleotide Phosphodiesterase Domain and a GGDEF Domain with ATPase Activity

The Functional Role of a Conserved Loop in EAL Domain-Based Cyclic di-GMP-Specific Phosphodiesterase