A non-canonical role: The Hippo pathway regulator MAP4K2 controls autophagy and cell survival upon energy stress.

In this issue, Seo et al.1 report a non-canonical function of the Hippo kinase MAP4K2 in energy stress response by regulating autophagy and cell survival, with relevance and therapeutic potential for head and neck cancer treatment.

Multilayered regulation of autophagy by the Atg1 kinase orchestrates spatial and temporal control of autophagosome formation.

Autophagy is a conserved intracellular degradation pathway exerting various cytoprotective and homeostatic functions by using de novo double-membrane vesicle (autophagosome) formation to target a wide range of cytoplasmic material for vacuolar/lysosomal degradation. The Atg1 kinase is one of its key regulators, coordinating a complex signaling program to orchestrate autophagosome formation. Combining in vitro reconstitution and cell-based approaches, we demonstrate that Atg1 is activated by lipidated Atg8 (Atg8-PE), stimulating substrate phosphorylation along the growing autophagosomal membrane. Atg1-dependent phosphorylation of Atg13 triggers Atg1 complex dissociation, enabling rapid turnover of Atg1 complex subunits at the pre-autophagosomal structure (PAS). Moreover, Atg1 recruitment by Atg8-PE self-regulates Atg8-PE levels in the growing autophagosomal membrane by phosphorylating and thus inhibiting the Atg8-specific E2 and E3. Our work uncovers the molecular basis for positive and negative feedback imposed by Atg1 and how opposing phosphorylation and dephosphorylation events underlie the spatiotemporal regulation of autophagy.

Structural basis for the subunit assembly of the anaphase-promoting complex.

The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.

Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor.

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ∼10 Šresolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

Substrate recognition in selective autophagy and the ubiquitin-proteasome system.

Dynamic protein turnover through regulated protein synthesis and degradation ensures cellular growth, proliferation, differentiation and adaptation. Eukaryotic cells utilize two mechanistically distinct but largely complementary systems - the 26S proteasome and the lysosome (or vacuole in yeast and plants) - to effectively target a wide range of proteins for degradation. The concerted action of the ubiquitination machinery and the 26S proteasome ensures the targeted and tightly regulated degradation of a subset of commonly short-lived cellular proteins. Autophagy is a distinct degradation pathway, which transports a highly heterogeneous set of cargos in dedicated vesicles, called autophagosomes, to the lysosome. There the cargo becomes degraded and its molecular building blocks are recycled. While general autophagy randomly engulfs portions of the cytosol, selective autophagy employs dedicated cargo adaptors to specifically enrich the forming autophagosomes for a certain type of cargo as a response to various intra- or extracellular signals. Selective autophagy targets a wide range of cargos including long-lived proteins and protein complexes, organelles, protein aggregates and even intracellular microbes. In this review we summarize available data on cargo recognition mechanisms operating in selective autophagy and the ubiquitin-proteasome system (UPS), and emphasize their differences and common themes. Moreover, we derive general regulatory principles underlying cargo recognition in selective autophagy, and describe the system-wide crosstalk between these two cellular protein degradation systems. This article is part of a Special Issue entitled: Ubiquitin-Proteasome System. Guest Editors: Thomas Sommer and Dieter H. Wolf.

Evaluation of the performance of a new compression system in patients with lymphoedema.

In the acute phase of lymphoedema, patients require comprehensive decongestive therapy (CDT), which includes skin care, an exercise regimen, manual lymphatic drainage (MLD) and regular bandaging. This study was established to determine the effectiveness of a new system of bandage therapy, the 3M™ Coban™ 2 compression system. In total, 24 patients were entered into the study (12 from UK and 12 from Canada) with a variety of clinical presentations. The mean age of the groups was 57·4 years, which varied from 26 to 79 years. Body mass index (BMI) averaged 38·9 kg/m(2), with a range from 22·7 to 67·5 kg/m(2). Of the total, eight were women with arm lymphoedema, the remainder being men and women with lymphoedema of the lower limb. All were considered to be in need of CDT. After 19 days, the reduction of limb volume was measured, which indicated a mean limb volume reduction of 1210 ml (95% confidence interval, CI, 780-1641, P < 0·001). Leg affected patients experienced greater reduction than arm affected patients (1596 ml versus 438 ml), although both groups experienced significant reduction in limb volumes (both P < 0·001). Mean percentage changes in limb volume were 14·9% and 16·1% for legs and arms, respectively. The Measure Your Medical Outcome Profile questionnaire indicated significant improvement in symptoms considered important by the patient (P < 0·0001), which also led to improvements in skin quality by reducing skin thickness and firmness. The Coban 2 compression system provides good oedema reduction in both arms and legs to reduce limb volume and improvements in symptoms associated with lymphoedema.

Autophagosome membrane expansion is mediated by the N-terminus and cis-membrane association of human ATG8s.

Autophagy is an essential catabolic pathway which sequesters and engulfs cytosolic substrates via autophagosomes, unique double-membraned structures. ATG8 proteins are ubiquitin-like proteins recruited to autophagosome membranes by lipidation at the C-terminus. ATG8s recruit substrates, such as p62, and play an important role in mediating autophagosome membrane expansion. However, the precise function of lipidated ATG8 in expansion remains obscure. Using a real-time in vitro lipidation assay, we revealed that the N-termini of lipidated human ATG8s (LC3B and GABARAP) are highly dynamic and interact with the membrane. Moreover, atomistic MD simulation and FRET assays indicate that N-termini of LC3B and GABARAP associate in cis on the membrane. By using non-tagged GABARAPs, we show that GABARAP N-terminus and its cis-membrane insertion are crucial to regulate the size of autophagosomes in cells irrespectively of p62 degradation. Our study provides fundamental molecular insights into autophagosome membrane expansion, revealing the critical and unique function of lipidated ATG8.

Structural mechanism for the selective phosphorylation of DNA-loaded MCM double hexamers by the Dbf4-dependent kinase.

Loading of the eukaryotic replicative helicase onto replication origins involves two MCM hexamers forming a double hexamer (DH) around duplex DNA. During S phase, helicase activation requires MCM phosphorylation by Dbf4-dependent kinase (DDK), comprising Cdc7 and Dbf4. DDK selectively phosphorylates loaded DHs, but how such fidelity is achieved is unknown. Here, we determine the cryogenic electron microscopy structure of Saccharomyces cerevisiae DDK in the act of phosphorylating a DH. DDK docks onto one MCM ring and phosphorylates the opposed ring. Truncation of the Dbf4 docking domain abrogates DH phosphorylation, yet Cdc7 kinase activity is unaffected. Late origin firing is blocked in response to DNA damage via Dbf4 phosphorylation by the Rad53 checkpoint kinase. DDK phosphorylation by Rad53 impairs DH phosphorylation by blockage of DDK binding to DHs, and also interferes with the Cdc7 active site. Our results explain the structural basis and regulation of the selective phosphorylation of DNA-loaded MCM DHs, which supports bidirectional replication.

Psychosocial burden and body mass index are associated with dermatology-related quality of life in psoriasis patients.

BACKGROUND: Scientific evidence indicates that inflammatory processes may be involved in the progression of both psoriasis and depression via elevated peripheral proinflammatory cytokines. OBJECTIVES: The aim of our study was to assess the association among psychological burden, depressive symptoms and proinflammatory mediators in psoriasis patients. MATERIALS AND METHODS: Forty psoriasis patients were recruited from the Department of Dermatology, University Hospital Essen. In addition to the Psoriasis Area and Severity Index (PASI), mental and physical health were explored using different questionnaires. Furthermore, proinflammatory cytokines were analysed. RESULTS: Patients in the high PASI group showed reduced Dermatology Life Quality Index (DLQI), higher body mass index (BMI), elevated CRP levels as well as impaired physical aspects of quality of life. Regression analyses revealed that somatic and anxiety symptoms accounted for more than 32% of the variance in DLQI, independent of PASI and cytokine levels. CONCLUSION: The data indicate somatic and anxiety symptoms, as well as BMI, to be closely linked to dermatology-related quality of life.

Methamphetamine withdrawal and the restoration of cognitive functions - a study over a course of 6 months abstinence.

A continuously rising consumption of methamphetamine (MA) has been suggested to be associated with increasing cognitive dysfunction. The objective of this study was to investigate associations between cognitive functions and gender, drug using patterns and treatment-attending profiles of recently abstinent MA users over the course of six months abstinence. Data were collected from 108 participants in two inpatient rehabilitation centers. The mean duration of MA use was 11.5 years. Interviews and cognitive tests (cognitrone, Stroop, TMT, nback) were performed right after the withdrawal and again after approx. six months of abstinence. Comparisons and explorative analyses between the groups (gender, primary MA/ multidrug users, early dropouts/ completers) regarding cognitive variables were performed. At baseline a significant decline in general neuropsychological functioning and attention/concentration after ongoing years of consumption were found. After a period of six months abstinence, cognitive performances remained stable or improved significantly for cognitrone percentile and cognitive flexibility. Normal cognitive functions were measured in former MA users after acute withdrawal which remained stable and partly improved in those patients who refrained from substance abuse over six months. Continued long-term MA intake was the only identified indicator of poorer cognitive performance. These results point towards a regain of cognitive performance in patients abstinent from MA.

Chlorpyrifos induces anxiety-like behavior in offspring rats exposed during pregnancy.

Chlorpyrifos is a pesticide, member of the organophosphate class, widely used in several countries to manage insect pests on many agricultural crops. Currently, chlorpyrifos health risks are being reevaluated due to possible adverse effects, especially on the central nervous system. The aim of this study was to investigate the possible action of this pesticide on the behaviors related to anxiety and depression of offspring rats exposed during pregnancy. Wistar rats were treated orally with chlorpyrifos (0.01, 0.1, 1 and 10mg/kg/day) on gestational days 14-20. Male offspring behavior was evaluated on post-natal days 21 and 70 by the elevated plus-maze test, open field test and forced swimming test. The results demonstrated that exposure to 0.1, 1 or 10mg/kg/day of chlorpyrifos could induce anxiogenic-like, but not depressive-like behavior at post-natal day 21, without causing fetal toxicity. This effect was reversed on post-natal day 70.

Comparative study of cold hyperalgesia and mechanical allodynia in two animal models of neuropathic pain: different etiologies and distinct pathophysiological mechanisms

Abstract Neuropathic pain (NP) affects more than 8% of the global population. The proposed action of the transient receptor potential ankyrin 1 (TRPA1) as a mechanosensor and the characterization of the transient receptor potential melastatin 8 (TRPM8) as a cold thermosensor raises the question of whether these receptors are implicated in NP. Our study aimed to evaluate the involvement of TRPA1 and TRPM8 in cold and mechanical signal transduction to obtain a comparative view in rat models of streptozotocin-induced diabetes (STZ) and chronic constriction injury of the sciatic nerve (CCI). The electronic von Frey test showed that STZ rats presented mechanical allodynia that was first evidenced on the 14th day after diabetes confirmation, and four days after CCI. This phenomenon was reduced by the intraplantar (ipl) administration of a TRPA1 receptor antagonist (HC-030031; 40 µL/300 µg/paw) in both NP models. Only CCI rats displayed cold hyperalgesia based on the cold plate test. The pharmacological blocking of TRPA1 through the injection of the antagonist attenuated cold hyperalgesia in this NP model. STZ animals showed a reduction in the number of flinches induced by the intraplantar injection of mustard oil (MO; TRPA1 agonist; 0.1%/50 µL/paw), or intraplantar injection of menthol (MT; TRPM8 agonist; 0.5% and 1%/50 µL/paw). The response induced by the ipl administration of MT (1%/50 µL/paw) was significantly different between the CCI and SHAM groups. Together, these data suggest a different pattern in nociceptive behavior associated with different models of NP, suggesting a variant involvement of TRPA1 and TRPM8 in both conditions

Effect of omega-3 polyunsaturated fatty acid treatment over mechanical allodynia and depressive-like behavior associated with experimental diabetes.

Neuropathic pain and depression are very common comorbidities in diabetic patients. As the pathophysiological mechanisms are very complex and multifactorial, current treatments are only symptomatic and often worsen the glucose control. Thus, the search for more effective treatments are extremely urgent. In this way, we aimed to investigate the effect of chronic treatment with fish oil (FO), a source of omega-3 polyunsaturated fatty acid, over the mechanical allodynia and in depressive-like behaviors in streptozotocin-diabetic rats. It was observed that the diabetic (DBT) animals, when compared to normoglycemic (NGL) animals, developed a significant mechanical allodynia since the second week after diabetes induction, peaking at fourth week which is completely prevented by FO treatment (0.5, 1 or 3g/kg). Moreover, DBT animals showed an increase of immobility frequency and a decrease of swimming and climbing frequencies in modified forced swimming test (MFST) since the second week after diabetes injection, lasting up at the 4th week. FO treatment (only at a dose of 3g/kg) significantly decreased the immobility frequency and increased the swimming frequency, but did not induce significant changes in the climbing frequency in DBT rats. Moreover, it was observed that DBT animals had significantly lower levels of BDNF in both hippocampus and pre frontal cortex when compared to NGL rats, which is completely prevented by FO treatment. In conclusion, our study demonstrates that FO treatment was able to prevent the mechanical allodynia and the depressive-like behaviors in DBT rats, which seems to be related to its capacity of BDNF level restoration.

Depression Associated with Diabetes: From Pathophysiology to Treatment.

Diabetes is a chronic and progressive syndrome commonly associated with several neuropsychiatric comorbities, of which depression is the most studied. The prevalence of depression is about two or three times higher in diabetic patients compared to the general population. It is believed that the diabetes - depression relation may be bidirectional, i.e., the depression can lead to diabetes and conversely diabetes could facilitate the emergence of depression. Depression is one of the most neglected symptoms in diabetic patients and is directly linked with lowering of quality of life. The treatment of depression in these patients is still quite ineffective and in many cases treatmentrefractory. Furthermore, some of the first choice drugs used to treat the depression affect the blood glucose control, aggravating the hyperglycemic state. These issues underscore the urgency in studies searching for new pharmacological targets for the treatment of depression associated with diabetes. For this, a better understanding of the pathophysiology that relates this comorbidity becomes critical. In this respect, this review will focus on some hypotheses that have been proposed to explain the mechanisms underlying depression associated with diabetes, highlighting the treatment options currently available and their limitations. Among these hypotheses, we will point out the hyperglycemia as a primary metabolic cause of the depression development, the involvement of the dysregulation of hypothalamic pituitary-adrenal (HPA) axis and of neurotransmitter systems, specially monoaminergic system. Besides, the role of oxidative stress, neuroinflammation and cell death, especially in hippocampus and prefrontal cortex, brain areas important for the mediation and modulation of emotional behavior will also be discussed. Finally, we will bring up the influence of the epigenetic regulation with respect to neuropsychiatric disorders.

Phytochemical and Antinociceptive, Anti-Inflammatory, and Antioxidant Studies of Smilax larvata (Smilacaceae).

The tea of aerial parts of Smilax larvata Griseb. (Smilacaceae) has been ethnopharmacologically used in Southern Brazil due to its anti-inflammatory action. In this study, ethanolic and organic extracts from aerial parts of S. larvata were phytochemically and pharmacologically characterized. The phytochemical analysis of EtOAc extract of S. larvata revealed the presence of three flavonoids, drabanemoroside, kaempferol 3-O-α-L-rhamnopyranosyl(1→2)-α-L-rhamnopyranoside, and kaempferol, the first two being isolated for the first time in this genus, two phenolic compounds p-hydroxybenzoic acid and p-coumaric acid, and alkaloids. In vitro assays demonstrated a potential antioxidant property of SLG. The treatment with SLG induced a significant reduction of the formalin-evoked flinches in rats, an effect reversed by opioid antagonist naloxone. Treatment with SLG also induced a significant increase in the hot plate latency and a decrease of intestinal motility by 45%. No effect was observed over nociceptive responses induced by a TRPA1 agonist mustard oil or over acetic acid-induced writhing in mice. Together, our data suggested that SLG has an in vivo antinociceptive effect, which seems to be associated with the opioid system activation. These findings support previous claims of medical use of Smilax larvata in the treatment of pain conditions.

Structural and kinetic analysis of the COP9-Signalosome activation and the cullin-RING ubiquitin ligase deneddylation cycle.

The COP9-Signalosome (CSN) regulates cullin-RING ubiquitin ligase (CRL) activity and assembly by cleaving Nedd8 from cullins. Free CSN is autoinhibited, and it remains unclear how it becomes activated. We combine structural and kinetic analyses to identify mechanisms that contribute to CSN activation and Nedd8 deconjugation. Both CSN and neddylated substrate undergo large conformational changes upon binding, with important roles played by the N-terminal domains of Csn2 and Csn4 and the RING domain of Rbx1 in enabling formation of a high affinity, fully active complex. The RING domain is crucial for deneddylation, and works in part through conformational changes involving insert-2 of Csn6. Nedd8 deconjugation and re-engagement of the active site zinc by the autoinhibitory Csn5 glutamate-104 diminish affinity for Cul1/Rbx1 by ~100-fold, resulting in its rapid ejection from the active site. Together, these mechanisms enable a dynamic deneddylation-disassembly cycle that promotes rapid remodeling of the cellular CRL network.

Translocon component Sec62 acts in endoplasmic reticulum turnover during stress recovery.

The endoplasmic reticulum (ER) is a site of protein biogenesis in eukaryotic cells. Perturbing ER homeostasis activates stress programs collectively called the unfolded protein response (UPR). The UPR enhances production of ER-resident chaperones and enzymes to reduce the burden of misfolded proteins. On resolution of ER stress, ill-defined, selective autophagic programs remove excess ER components. Here we identify Sec62, a constituent of the translocon complex regulating protein import in the mammalian ER, as an ER-resident autophagy receptor. Sec62 intervenes during recovery from ER stress to selectively deliver ER components to the autolysosomal system for clearance in a series of events that we name recovER-phagy. Sec62 contains a conserved LC3-interacting region in the C-terminal cytosolic domain that is required for its function in recovER-phagy, but is dispensable for its function in the protein translocation machinery. Our results identify Sec62 as a critical molecular component in maintenance and recovery of ER homeostasis.

Diabetic neuropathic pain: Physiopathology and treatment.

Diabetic neuropathy is a common complication of both type 1 and type 2 diabetes, which affects over 90% of the diabetic patients. Although pain is one of the main symptoms of diabetic neuropathy, its pathophysiological mechanisms are not yet fully known. It is widely accepted that the toxic effects of hyperglycemia play an important role in the development of this complication, but several other hypotheses have been postulated. The management of diabetic neuropathic pain consists basically in excluding other causes of painful peripheral neuropathy, improving glycemic control as a prophylactic therapy and using medications to alleviate pain. First line drugs for pain relief include anticonvulsants, such as pregabalin and gabapentin and antidepressants, especially those that act to inhibit the reuptake of serotonin and noradrenaline. In addition, there is experimental and clinical evidence that opioids can be helpful in pain control, mainly if associated with first line drugs. Other agents, including for topical application, such as capsaicin cream and lidocaine patches, have also been proposed to be useful as adjuvants in the control of diabetic neuropathic pain, but the clinical evidence is insufficient to support their use. In conclusion, a better understanding of the mechanisms underlying diabetic neuropathic pain will contribute to the search of new therapies, but also to the improvement of the guidelines to optimize pain control with the drugs currently available.

Lytic water dynamics reveal evolutionarily conserved mechanisms of ATP hydrolysis by TIP49 AAA+ ATPases.

Eukaryotic TIP49a (Pontin) and TIP49b (Reptin) AAA+ ATPases play essential roles in key cellular processes. How their weak ATPase activity contributes to their important functions remains largely unknown and difficult to analyze because of the divergent properties of TIP49a and TIP49b proteins and of their homo- and hetero-oligomeric assemblies. To circumvent these complexities, we have analyzed the single ancient TIP49 ortholog found in the archaeon Methanopyrus kandleri (mkTIP49). All-atom homology modeling and molecular dynamics simulations validated by biochemical assays reveal highly conserved organizational principles and identify key residues for ATP hydrolysis. An unanticipated crosstalk between Walker B and Sensor I motifs impacts the dynamics of water molecules and highlights a critical role of trans-acting aspartates in the lytic water activation step that is essential for the associative mechanism of ATP hydrolysis.

Peripheral antinociceptive effect of anandamide and drugs that affect the endocannabinoid system on the formalin test in normal and streptozotocin-diabetic rats.

Diabetes is often associated with painful neuropathy. The current treatments are symptomatic and ineffective. Cannabinoids have been proposed as promising drugs for chronic pain treatment and its antinociceptive effect has already been related in nerve injury models of neuropathic pain, but little has been investigated in painful diabetic neuropathy models. Thus, the current study aims to investigate the potential antinociceptive effect of drugs that alter endocannabinoid system when injected subcutaneously into the dorsal surface of the ipsilateral hind paw in chemical hyperalgesia induced by formalin in both normoglycemic (Ngl) and streptozotocin-diabetic (Dbt) rats. Diabetic rats exhibited exaggerated flinching behaviors during first and second phases of the formalin test, indicating the presence of hyperalgesia. AM404, an anandamide (AEA) re-uptake inhibitor, AEA (an agonist of CB1/CB2 receptors) or ACEA (a selective CB1 receptor agonist) induced antinociception in both phases of formalin test in Ngl and Dbt rats. In both groups, the antinociceptive effect of ACEA was prevented by AM251, a CB1 inverse agonist while the antinociceptive effect of AEA was prevented by AM251 or AM630, a CB2 receptor antagonist. In Ngl rats, the antinociceptive effect of AM404 was prevented by AM251 or capsazepine only during first phase of the formalin test while in Dbt rats, this effect was blocked by pretreatment with AM251 (both phases) or AM630 (second phase). Taken together, these results demonstrated broad-spectrum antinociceptive properties of cannabinoids in a model of painful diabetic neuropathy. Peripheral activation of both cannabinoid receptors seems to mediate the antinociceptive effect of exogenous or endogenous anandamide.