Identification of a bacterial type III effector family with G protein mimicry functions

Many bacterial pathogens use the type III secretion system to inject "effector" proteins into host cells. Here, we report the identification of a 24 member effector protein family found in pathogens including Salmonella, Shigella, and enteropathogenic E. coli. Members of this family subvert host cell function by mimicking the signaling properties of Ras-like GTPases. The effector IpgB2 stimulates cellular responses analogous to GTP-active RhoA, whereas IpgB1 and Map function as the active forms of Rac1 and Cdc42, respectively. These effectors do not bind guanine nucleotides or have sequences corresponding the conserved GTPase domain, suggesting that they are functional but not structural mimics. However, several of these effectors harbor intracellular targeting sequences that contribute to their signaling specificities. The activities of IpgB2, IpgB1, and Map are dependent on an invariant WxxxE motif found in numerous effectors leading to the speculation that they all function by a similar molecular mechanism.     

Signaling specificity by Ras family GTPases is determined by the full spectrum of effectors they regulate

Ras family GTPases (RFGs) regulate signaling pathways that control multiple biological processes. How signaling specificity among the closely related family members is achieved is poorly understood. We have taken a proteomics approach to signaling by RFGs, and we have analyzed interactions of a panel of RFGs with a comprehensive group of known and potential effectors. We have found remarkable differences in the ability of RFGs to regulate the various isoforms of known effector families. We have also identified several proteins as novel effectors of RFGs with differential binding specificities to the various RFGs. We propose that specificity among RFGs is achieved by the differential regulation of combinations of effector families as well as by the selective regulation of different isoforms within an effector family. An understanding of this new level of complexity in the signaling pathways regulated by RFGs is necessary to understand how they carry out their many cellular functions. It will also likely have critical implications in the treatment of human diseases such as cancer.     

Beyond ATM: the protein kinase landscape of the DNA damage response

The DNA of all organisms is constantly subjected to damaging agents, both exogenous and endogenous. One extremely harmful lesion is the double-strand break (DSB), which activates a massive signaling network - the DNA damage response (DDR). The chief activator of the DSB response is the ATM protein kinase, which phosphorylates numerous key players in its various branches. Recent phosphoproteomic screens have extended the scope of damage-induced phosphorylations beyond the direct ATM substrates. We review the evidence for the involvement of numerous other protein kinases in the DDR, obtained from documentation of specific pathways as well as high-throughput screens. The emerging picture of the protein phosphorylation landscape in the DDR broadens the current view on the role of this protein modification in the maintenance of genomic stability. Extensive cross-talk between many of these protein kinases forms an interlaced signaling network that spans numerous cellular processes. Versatile protein kinases in this network affect pathways that are different from those they have been identified with to date. The DDR appears to be one of the most extensive signaling responses to cellular stimuli.     

Structural biology of G protein‐coupled receptor signaling complexes

G protein‐coupled receptors (GPCRs) constitute the largest family of cell surface receptors that mediate numerous cell signaling pathways, and are targets of more than one‐third of clinical drugs. Thanks to the advancement of novel structural biology technologies, high‐resolution structures of GPCRs in complex with their signaling transducers, including G‐protein and arrestin, have been determined. These 3D complex structures have significantly improved our understanding of the molecular mechanism of GPCR signaling and provided a structural basis for signaling‐biased drug discovery targeting GPCRs. Here we summarize structural studies of GPCR signaling complexes with G protein and arrestin using rhodopsin as a model system, and highlight the key features of GPCR conformational states in biased signaling including the sequence motifs of receptor TM6 that determine selective coupling of G proteins, and the phosphorylation codes of GPCRs for arrestin recruitment. We envision the future of GPCR structural biology not only to solve more high‐resolution complex structures but also to show stepwise GPCR signaling complex assembly and disassembly and dynamic process of GPCR signal transduction.     

The role of TRIM25 in the occurrence and development of cancers and inflammatory diseases

The tripartite motif (TRIM) family proteins are a group of proteins involved in different signaling pathways. The changes in the expression regulation, function, and signaling of this protein family are associated with the occurrence and progression of a wide range of disorders. Given the importance of these proteins in pathogenesis, they can be considered as potential therapeutic targets for many diseases. TRIM25, as an E3-ubiquitin ligase, is involved in the development of various diseases and cellular mechanisms, including antiviral innate immunity and cell proliferation. The clinical studies conducted on restricting the function of this protein have reached promising results that can be further evaluated in the future. Here, we review the regulation of TRIM25 and its function in different diseases and signaling pathways, especially the retinoic acid-inducible gene-I (RIG-I) signaling which prompts many kinds of cancers and inflammatory disorders.     

The multifaceted roles of the HORMA domain in cellular signaling

The HORMA domain is a multifunctional protein–protein interaction module found in diverse eukaryotic signaling pathways including the spindle assembly checkpoint, numerous DNA recombination/repair pathways, and the initiation of autophagy. In all of these pathways, HORMA domain proteins occupy key signaling junctures and function through the controlled assembly and disassembly of signaling complexes using a stereotypical “safety belt” peptide interaction mechanism. A recent explosion of structural and functional work has shed new light on these proteins, illustrating how strikingly similar structural mechanisms give rise to radically different functional outcomes in each family of HORMA domain proteins.     

The Nod-like receptor (NLR) family: a tale of similarities and differences

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.     

Structural basis of the Rho GTPase signaling

Small GTPases of the Rho family serve as conformational switches in a wide variety of signal transduction pathways that regulate diverse cellular functions. The GTP-bound forms of Rho GTPases are capable of interacting with downstream effectors that control cytoskeletal rearrangements. Regulators that stimulate nucleotide exchange, the hydrolytic cycle and distribution between the membrane and cytosol control the switch. Detailed pictures of Rho GTPase switching, effector recognition and regulation by regulators have emerged from recent structural investigations. These include the most extensively studied Rho GTPases, RhoA, Rac1, 2 and Cdc42, and their complexes with effectors and regulators. These studies have revealed the general diversity of effector and regulator structures, and in particular the structural features concerning the specific interactions involved in Rho effector recognition and regulator interactions with Rho GTPase. These findings provide a critical insight into the nature of Rho GTPase activity and consequently allow for a detailed manipulation of signaling pathways mediated by these proteins.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

Mutations in the Effector Domain of RhoV GTPase Impair Its Binding to Pak1 Protein Kinase

Atypical RhoV GTPase (Chp/Wrch-2) is a member of the human Rho GTPase family, which belongs to the superfamily of Ras-related small GTPases. The biological functions of RhoV, regulation of its activity, and mechanisms of its action remain largely unexplored. Rho GTPases regulate a wide range of cellular processes by interacting with protein targets called effectors. Several putative RhoV effectors have been identified, including protein kinases of the Pak (p21-activated kinase) family: Pak1, Pak2, Pak4, and Pak6. RhoV GTPase activates Pak1 protein kinase and simultaneously induces its ubiquitin-dependent degradation. Pak1 regulates E-cadherin localization at adherens junctions downstream of RhoV during gastrulation in fish. The effector domain of RhoV mediates its binding to the CRIB (Cdc42/Rac1 interactive binding) motif in the N-terminal p21-binding domain (PBD) of Pak6 protein kinase. The role of the RhoV effector domain in mediating interaction with Pak1 has not been studied. This study has identified mutations in the effector domain of RhoV GTPase (Y60K, T63A, L65A, and D66A) that impair its interaction with Pak1 in the GST-PAK-PBD pull-down assay and coimmunoprecipitation. Our results suggest that the effector domain of RhoV mediates its binding to Pak1, complementing the current view of the molecular basics of RhoV binding to effectors of the Pak family. These data lay the basis for further studies on the role of Pak1 in RhoV-activated signaling pathways and cellular processes.     

Functions of the Yersinia effector proteins in inhibiting host immune responses

The invasion strategies used by Yersinia species involve the 'hijacking' of host cellular signaling pathways, often involving microbial gene products that mimic the functions of the cellular proteins. Yersinia uses a type III secretion system to inject these microbial gene products, referred to as Yersinia effector proteins, into the host cytosol. Yersinia effector proteins can inhibit the host immune system through a diverse array of mechanisms including inhibition of the inflammatory response by interfering with cytokine production, inhibition of phagocytosis by disrupting the actin cytoskeleton, induction of apoptosis in macrophages and through the formation of novel signaling complexes.     

Biological functions of the small leucine-rich proteoglycans: from genetics to signal transduction

The small leucine-rich proteoglycan (SLRP) family has significantly expanded in the past decade to now encompass five discrete classes, grouped by common structural and functional properties. Some of these gene products are not classical proteoglycans, whereas others have new and unique features. In addition to being structural proteins, SLRPs constitute a network of signal regulation: being mostly extracellular, they are upstream of multiple signaling cascades. They affect intracellular phosphorylation, a major conduit of information for cellular responses, and modulate distinct pathways, including those driven by bone morphogenetic protein/transforming growth factor beta superfamily members, receptor tyrosine kinases such as ErbB family members and the insulin-like growth factor I receptor, and Toll-like receptors. The wealth of mechanistic insights into the molecular and cellular functions of SLRPs has revealed both the sophistication of this family of regulatory proteins and the challenges that remain in uncovering the totality of their functions. This review is focused on novel biological functions of SLRPs with special emphasis on their protein cores, newly described genetic diseases, and signaling events in which SLRPs play key functions.     

Ceramide and other sphingolipids in cellular responses

Formerly considered to serve only as structural components, sphingolipids are emerging as an important group of signaling molecules involved in many cellular events, including cell growth, senescence, meiotic maturation, and cell death. They are also implicated in functions such as inflammation and the responses to heat shock and genotoxic stress. Defects in the metabolism of sphingolipids are related to various genetic disorders, and sphingolipids have the potential to serve as therapeutic agents for human diseases such as colon cancer and viral or bacterial infections. The best-studied member of this family, ceramide, which also serves as the structural back-bone for other sphingolipids, is an important mediator in multiple cellular signaling pathways. The metabolism and functions of sphingolipids are discussed in this review, with a focus on ceramide regulation in various cellular responses.     

Specificity of G protein-RGS protein recognition is regulated by affinity adapters

RGS proteins regulate the duration of cell signaling by modulating the lifetime of activated G proteins. The specificity of RGS-G protein mutual recognition is critical for meeting unique timing requirements of numerous G protein-mediated pathways. Our study of two splice isoforms of RGS9 expressed in different types of neurons revealed a novel mechanism whereby this specificity is determined by specialized protein domains or subunits acting as affinity adapters. The long RGS9 isoform contains a C-terminal domain that provides high-affinity interaction with its target G protein. The lack of this domain in the short RGS9 isoform is compensated by the action of a G protein effector subunit that is structurally similar to this C-terminal domain. This allows the short isoform to specifically target the complex between the G protein and its effector. Thus, the specific timing needs of different signaling pathways can be accommodated by affinity adapters positioned at various pathway components.     

Role of cAMP and phosphodiesterase signaling in liver health and disease

Liver disease is a significant health problem worldwide with mortality reaching around 2 million deaths a year. Non-alcoholic fatty liver disease (NAFLD) and alcoholic liver disease (ALD) are the major causes of chronic liver disease. Pathologically, NAFLD and ALD share similar patterns of hepatic disorders ranging from simple steatosis to steatohepatitis, fibrosis and cirrhosis. It is becoming increasingly important to identify new pharmacological targets, given that there is no FDA-approved therapy yet for either NAFLD or ALD. Since the evolution of liver diseases is a multifactorial process, several mechanisms involving parenchymal and non-parenchymal hepatic cells contribute to the initiation and progression of liver pathologies. Moreover, certain protective molecular pathways become repressed during liver injury including signaling pathways such as the cyclic adenosine monophosphate (cAMP) pathway. cAMP, a key second messenger molecule, regulates various cellular functions including lipid metabolism, inflammation, cell differentiation and injury by affecting gene/protein expression and function. This review addresses the current understanding of the role of cAMP metabolism and consequent cAMP signaling pathway(s) in the context of liver health and disease. The cAMP pathway is extremely sophisticated and complex with specific cellular functions dictated by numerous factors such abundance, localization and degradation by phosphodiesterases (PDEs). Furthermore, because of the distinct yet divergent roles of both of its effector molecules, the cAMP pathway is extensively targeted in liver injury to modify its role from physiological to therapeutic, depending on the hepatic condition. This review also examines the behavior of the cAMP-dependent pathway in NAFLD, ALD and in other liver diseases and focuses on PDE inhibition as an excellent therapeutic target in these conditions.