A sterol-binding protein integrates endosomal lipid metabolism with TOR signaling and nitrogen sensing.

Kes1, and other oxysterol-binding protein superfamily members, are involved in membrane and lipid trafficking through trans-Golgi network (TGN) and endosomal systems. We demonstrate that Kes1 represents a sterol-regulated antagonist of TGN/endosomal phosphatidylinositol-4-phosphate signaling. This regulation modulates TOR activation by amino acids and dampens gene expression driven by Gcn4, the primary transcriptional activator of the general amino acid control regulon. Kes1-mediated repression of Gcn4 transcription factor activity is characterized by nonproductive Gcn4 binding to its target sequences, involves TGN/endosome-derived sphingolipid signaling, and requires activity of the cyclin-dependent kinase 8 (CDK8) module of the enigmatic "large Mediator" complex. These data describe a pathway by which Kes1 integrates lipid metabolism with TORC1 signaling and nitrogen sensing.

Predictors of recurrence and long-term patient reported outcomes following surgical repair of anal fistula, a retrospective analysis.

PURPOSE: Surgery for anal fistulas can result in devastating complications, including reoperations and fecal incontinence. There is limited contemporary evidence comparing outcomes since the adoption of the ligation of intersphincteric fistula tract procedure into mainstream practice. The purpose of this study is to compare recurrence rates and long-term outcomes of anal fistula following repair. METHODS: Data was collected from the electronic medical records or patient reported outcomes from patients aged 18 or older with a primary or recurrent cryptoglandular anal fistula. Primary outcome was recurrence defined as the identification of at least one fistula os or a high clinical suspicion of anal fistula. Secondary outcomes included fecal incontinence and postoperative quality of life. RESULTS: A total of 171 patients underwent definitive surgical repairs for their anal fistula. So 66.5% had a simple fistula, and 33.5% had a complex fistula. Of the 171 patients, 12.5% had a recurrence. The recurrence rates were 5.9% for simple fistula and 25.4% for complex fistula. Predictors of recurrence included diabetes mellitus, history of anorectal abscess, complex fistula, and sphincter sparing surgery. LIFT or plug/biologic procedures were both associated with a 50% or greater recurrence rate. No significant differences were found in fecal incontinence or associated quality of life between sphincter sparing or non-sphincter sparing surgical resections. CONCLUSION: The study provides insights into the long-term outcomes of surgical repair for anal fistula. We demonstrate that sphincter sparing operations are associated with increased recurrence, meanwhile, non-sphincter sparing surgeries did not increase the risk of fecal incontinence or worsen quality of life.

Decompressive Cranial Vault Remodeling in Osteosclerotic Robinow Syndrome.

We present a novel application of endocranial burr contouring for cranial vault expansion as a surgical adjunct during decompressive craniectomy in patients with cranial osteosclerosis. A 16-year-old female with osteosclerotic Robinow syndrome complicated by slit ventricle syndrome presented with refractory intracranial hypertension following external ventricular drain placement. Symptoms included severe headaches and altered mental status. Given the severe intracranial volume restriction secondary to massive calvarial thickening (2.5 cm), the patient was taken to the operating room for urgent surgical decompression. After frontal and parietal craniectomy, burr and osteotome contouring were used to remove two-thirds of the endocranial calvarial bone flap thickness resulting in a 9% cranial vault expansion while preserving an overall normal head size. There were no immediate postoperative complications. At over 3 years postoperatively, the patient had reduced headaches, maintained adequate shunt function, and has not required further vault reconstruction.

The cAMP-dependent protein kinase signaling pathway is a key regulator of P body foci formation.

In response to stress, eukaryotic cells accumulate mRNAs and proteins at discrete sites, or foci, in the cytoplasm. However, the mechanisms regulating foci formation, and the biological function of the larger ribonucleoprotein (RNP) assemblies, remain poorly understood. Here, we show that the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae is a key regulator of the assembly of processing bodies (P bodies), an RNP complex implicated in mRNA processing and translation. The data suggest that PKA specifically inhibits the formation of the larger P body aggregates by directly phosphorylating Pat1, a conserved constituent of these foci that functions as a scaffold during the assembly process. Finally, we present evidence indicating that P body foci are required for the long-term survival of stationary phase cells. This work therefore highlights the general relevance of RNP foci in quiescent cells, and provides a framework for the study of the many RNP assemblies that form in eukaryotic cells.

Neuregulin and dopamine modulation of hippocampal gamma oscillations is dependent on dopamine D4 receptors.

The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations--a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1-induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.

An Atg13 protein-mediated self-association of the Atg1 protein kinase is important for the induction of autophagy.

Autophagy pathways in eukaryotic cells mediate the turnover of a diverse set of cytoplasmic components, including damaged organelles and abnormal protein aggregates. Autophagy-mediated degradation is highly regulated, and defects in these pathways have been linked to a number of human disorders. The Atg1 protein kinase appears to be a key site of this control and is targeted by multiple signaling pathways to ensure the appropriate autophagic response to changing environmental conditions. Despite the importance of this kinase, relatively little is known about the molecular details of Atg1 activation. In this study we show that Atg13, an evolutionarily conserved regulator of Atg1, promotes the formation of a specific Atg1 self-interaction in the budding yeast, Saccharomyces cerevisiae. The appearance of this Atg1-Atg1 complex is correlated with the induction of autophagy, and conditions that disrupt this complex result in diminished levels of both autophagy and Atg1 kinase activity. Moreover, the addition of a heterologous dimerization domain to Atg1 resulted in elevated kinase activity both in vivo and in vitro. The formation of this complex appears to be an important prerequisite for the subsequent autophosphorylation of Thr-226 in the Atg1 activation loop. Previous work indicates that this modification is necessary and perhaps sufficient for Atg1 kinase activity. Interestingly, this Atg1 self-association does not require Atg17, suggesting that this second conserved regulator might activate Atg1 in a manner mechanistically distinct from that of Atg13. In all, this work suggests a model whereby this self-association stimulates the autophosphorylation of Atg1 within its activation loop.

The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy.

Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.

A distinct P-body-like granule is induced in response to the disruption of microtubule integrity in Saccharomyces cerevisiae.

The Processing-body is a conserved membraneless organelle that has been implicated in the storage and/or decay of mRNAs. Although Processing-bodies have been shown to be induced by a variety of conditions, the mechanisms controlling their assembly and their precise physiological roles in eukaryotic cells are still being worked out. In this study, we find that a distinct subtype of Processing-body is induced in response to conditions that disrupt microtubule integrity in the budding yeast, Saccharomyces cerevisiae. For example, treatment with the microtubule-destabilizing agent, benomyl, led to the induction of these novel ribonucleoprotein granules. A link to microtubules had been noted previously and the observations here extend our understanding by demonstrating that the induced foci differ from traditional P-bodies in a number of significant ways. These include differences in overall granule morphology, protein composition, and the manner in which their induction is regulated. Of particular note, several key Processing-body constituents are absent from these benomyl-induced granules, including the Pat1 protein that is normally required for efficient Processing-body assembly. However, these novel ribonucleoprotein structures still contain many known Processing-body proteins and exhibit similar hallmarks of a liquid-like compartment. In all, the data suggest that the disruption of microtubule integrity leads to the formation of a novel type of Processing-body granule that may have distinct biological activities in the cell. Future work will aim to identify the biological activities of these benomyl-induced granules and to determine, in turn, whether these Processing-body-like granules have any role in the regulation of microtubule dynamics.

Distal recognition sites in substrates are required for efficient phosphorylation by the cAMP-dependent protein kinase.

Protein kinases are important mediators of signal transduction in eukaryotic cells, and identifying the substrates of these enzymes is essential for a complete understanding of most signaling networks. In this report, novel substrate-binding variants of the cAMP-dependent protein kinase (PKA) were used to identify substrate domains required for efficient phosphorylation in vivo. Most wild-type protein kinases, including PKA, interact only transiently with their substrates. The substrate domains identified were distal to the sites of phosphorylation and were found to interact with a C-terminal region of PKA that was itself removed from the active site. Only a small set of PKA alterations resulted in a stable association with substrates, and the identified residues were clustered together within the hydrophobic core of this enzyme. Interestingly, these residues stretched from the active site of the enzyme to the C-terminal substrate-binding domain identified here. This spatial organization is conserved among the entire eukaryotic protein kinase family, and alteration of these residues in a second, unrelated protein kinase also resulted in a stable association with substrates. In all, this study identified distal sites in PKA substrates that are important for recognition by this enzyme and suggests that the interaction of these domains with PKA might influence specific aspects of substrate binding and/or release.

Insights into the Role of P-Bodies and Stress Granules in Protein Quality Control.

The eukaryotic cell is highly compartmentalized, and contains a variety of both membrane-bound and membraneless organelles. The latter include the cytoplasmic ribonucleoprotein (RNP) granules, known as the processing body (P-body) and the stress granule. These RNP structures are thought to be involved in the storage of particular mRNAs during periods of stress. Here, we find that a mutant lacking both P-bodies and stress granules exhibits phenotypes suggesting that these structures also have a role in the maintenance of protein homeostasis. In particular, there was an increased occurrence of specific protein quality control (PQC) compartments in this mutant, an observation that is consistent with there being an elevated level of protein misfolding. These compartments normally house soluble misfolded proteins and allow the cell to sequester these polypeptides away from the remaining cellular milieu. Moreover, specific proteins that are normally targeted to both P-bodies and stress granules were found to instead associate with these PQC compartments in this granuleless mutant. This observation is interesting as our data indicate that this association occurs specifically in cells that have been subjected to an elevated level of proteotoxic stress. Altogether, the results here are consistent with P-bodies and stress granules having a role in normal protein homeostasis in eukaryotic cells.

P-Body Localization of the Hrr25/Casein Kinase 1 Protein Kinase Is Required for the Completion of Meiosis.

P-bodies are liquid droplet-like compartments that lack a limiting membrane and are present in many eukaryotic cells. These structures contain specific sets of proteins and mRNAs at concentrations higher than that in the surrounding environment. Although highly conserved, the normal physiological roles of these ribonucleoprotein (RNP) granules remain poorly defined. Here, we report that P-bodies are required for the efficient completion of meiosis in the budding yeast Saccharomyces cerevisiae P-bodies were found to be present during all phases of the meiotic program and to provide protection for the Hrr25/CK1 protein kinase, a key regulator of this developmental process. A failure to associate with these RNP granules resulted in diminished levels of Hrr25 and an ensuing inability to complete meiosis. This work therefore identifies a novel function for these RNP granules and indicates how protein recruitment to these structures can have a significant impact on eukaryotic cell biology.

Using substrate-binding variants of the cAMP-dependent protein kinase to identify novel targets and a kinase domain important for substrate interactions in Saccharomyces cerevisiae.

Protein kinases mediate much of the signal transduction in eukaryotic cells and defects in kinase function are associated with a variety of human diseases. To understand and correct these defects, we will need to identify the physiologically relevant substrates of these enzymes. The work presented here describes a novel approach to this identification process for the cAMP-dependent protein kinase (PKA) in Saccharomyces cerevisiae. This approach takes advantage of two catalytically inactive PKA variants, Tpk1K336A/H338A and Tpk1R324A, that exhibit a stable binding to their substrates. Most protein kinases, including the wild-type PKA, associate with substrates with a relatively low affinity. The binding observed here was specific to substrates and was dependent upon PKA residues known to be important for interactions with peptide substrates. The general utility of this approach was demonstrated by the ability to identify both previously described and novel PKA substrates in S. cerevisiae. Interestingly, the positions of the residues altered in these variants implicated a particular region within the PKA kinase domain, corresponding to subdomain XI, in the binding and/or release of protein substrates. Moreover, the high conservation of the residues altered and, in particular, the invariant nature of the R324 position suggest that this approach might be generally applicable to other protein kinases.

Stationary phase in yeast.

Eukaryotic cell proliferation is controlled by specific growth factors and the availability of essential nutrients. If either of these signals is lacking, cells may enter into a specialized nondividing resting state, known as stationary phase or G(0). The entry into such resting states is typically accompanied by a dramatic decrease in the overall growth rate and an increased resistance to a variety of environmental stresses. Since most cells spend most of their life in these quiescent states, it is important that we develop a full understanding of the biology of the stationary phase/G(0) cell. This knowledge would provide important insights into the control of two of the most fundamental aspects of eukaryotic cell biology: cell proliferation and long-term cell survival. This review will discuss some recent advances in our understanding of the stationary phase of growth in the budding yeast, Saccharomyces cerevisiae.

A Hybrid-Body Containing Constituents of Both P-Bodies and Stress Granules Forms in Response to Hypoosmotic Stress in Saccharomyces cerevisiae.

The cytoplasm of the eukaryotic cell is a highly compartmentalized space that contains a variety of ribonucleoprotein (RNP) granules in addition to its complement of membrane-bound organelles. These RNP granules contain specific sets of proteins and mRNAs and form in response to particular environmental and developmental stimuli. Two of the better-characterized of these RNP structures are the stress granule and Processing-body (P-body) that have been conserved from yeast to humans. In this report, we examined the cues regulating stress granule assembly and the relationship between stress granule and P-body foci. These two RNP structures are generally thought to be independent entities in eukaryotic cells. However, we found here that stress granule and P-body proteins were localized to a common or merged granule specifically in response to a hypoosmotic stress. Interestingly, these hybrid-bodies were found to be transient structures that were resolved with time into separate P-body and stress granule foci. In all, these data suggest that the identity of an RNP granule is not absolute and that it can vary depending upon the nature of the induction conditions. Since the activities of a granule are likely influenced by its protein constituency, these observations are consistent with the possibility of RNP granules having distinct functions in different cellular contexts.

The Catalytic Activity of the Ubp3 Deubiquitinating Protease Is Required for Efficient Stress Granule Assembly in Saccharomyces cerevisiae.

The interior of the eukaryotic cell is a highly compartmentalized space containing both membrane-bound organelles and the recently identified nonmembranous ribonucleoprotein (RNP) granules. This study examines in Saccharomyces cerevisiae the assembly of one conserved type of the latter compartment, known as the stress granule. Stress granules form in response to particular environmental cues and have been linked to a variety of human diseases, including amyotrophic lateral sclerosis. To further our understanding of these structures, a candidate genetic screen was employed to identify regulators of stress granule assembly in quiescent cells. These studies identified a ubiquitin-specific protease, Ubp3, as having an essential role in the assembly of these RNP granules. This function was not shared by other members of the Ubp protease family and required Ubp3 catalytic activity as well as its interaction with the cofactor Bre5. Interestingly, the loss of stress granules was correlated with a decrease in the long-term survival of stationary-phase cells. This phenotype is similar to that observed in mutants defective for the formation of a related RNP complex, the Processing body. Altogether, these observations raise the interesting possibility of a general role for these types of cytoplasmic RNP granules in the survival of G0-like resting cells.

The Activity-Dependent Regulation of Protein Kinase Stability by the Localization to P-Bodies.

The eukaryotic cytoplasm contains a variety of ribonucleoprotein (RNP) granules in addition to the better-understood membrane-bound organelles. These granules form in response to specific stress conditions and contain a number of signaling molecules important for the control of cell growth and survival. However, relatively little is known about the mechanisms responsible for, and the ultimate consequences of, this protein localization. Here, we show that the Hrr25/CK1δ protein kinase is recruited to cytoplasmic processing bodies (P-bodies) in an evolutionarily conserved manner. This recruitment requires Hrr25 kinase activity and the Dcp2 decapping enzyme, a core constituent of these RNP granules. Interestingly, the data indicate that this localization sequesters active Hrr25 away from the remainder of the cytoplasm and thereby shields this enzyme from the degradation machinery during these periods of stress. Altogether, this work illustrates how the presence within an RNP granule can alter the ultimate fate of the localized protein.

The Ras/PKA signaling pathway may control RNA polymerase II elongation via the Spt4p/Spt5p complex in Saccharomyces cerevisiae.

The Ras signaling pathway in Saccharomyces cerevisiae controls cell growth via the cAMP-dependent protein kinase, PKA. Recent work has indicated that these effects on growth are due, in part, to the regulation of activities associated with the C-terminal domain (CTD) of the largest subunit of RNA polymerase II. However, the precise target of these Ras effects has remained unknown. This study suggests that Ras/PKA activity regulates the elongation step of the RNA polymerase II transcription process. Several lines of evidence indicate that Spt5p in the Spt4p/Spt5p elongation factor is the likely target of this control. First, the growth of spt4 and spt5 mutants was found to be very sensitive to changes in Ras/PKA signaling activity. Second, mutants with elevated levels of Ras activity shared a number of specific phenotypes with spt5 mutants and vice versa. Finally, Spt5p was efficiently phosphorylated by PKA in vitro. Altogether, the data suggest that the Ras/PKA pathway might be directly targeting a component of the elongating polymerase complex and that this regulation is important for the normal control of yeast cell growth. These data point out the interesting possibility that signal transduction pathways might directly influence the elongation step of RNA polymerase II transcription.

Protein kinases are associated with multiple, distinct cytoplasmic granules in quiescent yeast cells.

The cytoplasm of the eukaryotic cell is subdivided into distinct functional domains by the presence of a variety of membrane-bound organelles. The remaining aqueous space may be further partitioned by the regulated assembly of discrete ribonucleoprotein (RNP) complexes that contain particular proteins and messenger RNAs. These RNP granules are conserved structures whose importance is highlighted by studies linking them to human disorders like amyotrophic lateral sclerosis. However, relatively little is known about the diversity, composition, and physiological roles of these cytoplasmic structures. To begin to address these issues, we examined the cytoplasmic granules formed by a key set of signaling molecules, the protein kinases of the budding yeast Saccharomyces cerevisiae. Interestingly, a significant fraction of these proteins, almost 20%, was recruited to cytoplasmic foci specifically as cells entered into the G0-like quiescent state, stationary phase. Colocalization studies demonstrated that these foci corresponded to eight different granules, including four that had not been reported previously. All of these granules were found to rapidly disassemble upon the resumption of growth, and the presence of each was correlated with cell viability in the quiescent cultures. Finally, this work also identified new constituents of known RNP granules, including the well-characterized processing body and stress granule. The composition of these latter structures is therefore more varied than previously thought and could be an indicator of additional biological activities being associated with these complexes. Altogether, these observations indicate that quiescent yeast cells contain multiple distinct cytoplasmic granules that may make important contributions to their long-term survival.

Processing body and stress granule assembly occur by independent and differentially regulated pathways in Saccharomyces cerevisiae.

A variety of ribonucleoprotein (RNP) granules form in eukaryotic cells to regulate the translation, decay, and localization of the encapsulated messenger RNA (mRNAs). The work here examined the assembly and function of two highly conserved RNP structures, the processing body (P body) and the stress granule, in the yeast Saccharomyces cerevisiae. These granules are induced by similar stress conditions and contain translationally repressed mRNAs and a partially overlapping set of protein constituents. However, despite these similarities, the data indicate that these RNP complexes are independently assembled and that this assembly is controlled by different signaling pathways. In particular, the cAMP-dependent protein kinase (PKA) was found to control P body formation under all conditions examined. In contrast, the assembly of stress granules was not affected by changes in either PKA or TORC1 signalling activity. Both of these RNP granules were also detected in stationary-phase cells, but each appears at a distinct time. P bodies were formed prior to stationary-phase arrest, and the data suggest that these foci are important for the long-term survival of these quiescent cells. Stress granules, on the other hand, were not assembled until after the cells had entered into the stationary phase of growth and their appearance could therefore serve as a specific marker for the entry into this quiescent state. In all, the results here provide a framework for understanding the assembly of these RNP complexes and suggest that these structures have distinct but important activities in quiescent cells.