Functional Characterization of Interaction Partners of the Co-Chaperone BAG3

Protein homeostasis (proteostasis) describes the cellular balance between protein synthesis, function and degradation. The stress inducible co-chaperone BAG3 is a key factor in proteostasis, in particular under mechanical stress. In cooperation with the actin binding protein Synaptopodin 2 (SYNPO2), BAG3 is a central element of filamin (an actin crosslinking protein) homeostasis in muscle cells and immune cells. Here BAG3 is involved in the degradation of defective filamin via chaperone assisted selective autophagy (CASA), as well as in the induction of filamin transcription via the Hippo pathway. SYNPO2 can directly bind to the WW-domain of BAG3 and through its N-terminal PDZ-domain SYNPO2 is enabled to recruit complexes mediating the formation of autophagosomes. SYNPO2 belongs to the SYNPO family of actin binding proteins. Four isoforms of SYNPO2 exist, of which three contain an N-terminal PDZ-domain, whereas all four isoforms have a centrally located proline-rich motif (PPXY-motif). SYNPO2 was identified to interact with BAG3 via its PPXY-motif in a peptde screen for novel binding partners of the BAG3 WWdomain. Another SYNPO family member, Synaptopodin (SYNPO), was also identified in this peptide screen. SYNPO has three isoforms of which all contain two subsequent PPXYmotifs. Furthermore, SYNPO has been described to interact with the PDZ-domain containing protein MAGI-1 (Membrane Associated Guanylate Kinase Inverted 1). In this work SYNPO could be confirmed as novel BAG3 binding partner. Because SYNPO2 is not expressed ubiquitously in all cell types, it should be clarified whether SYNPO, together with BAG3, is similarly involved in autophagic protein degradation as shown for SYNPO2. In HeLa cells, which do not express SYNPO2, SYNPO turnover was monitored in the context of inhibition of the proteasome or autophagy. In contrast to SYNPO2, which is rapidly degraded via autophagy upon inhibition of the proteasome or mechanical stress, SYNPO protein levels remained stable upon inhibiton of the proteasome or autophagy. Furthermore, no association of SYNPO with autophagic marker proteins such as LC3 or p62 could be observed in HeLa cells. These findings imply, that SYNPO functions differently from SYNPO2 under the chosen experimental conditions. Challenging the cells with increased cellular stress to enhance the induction of BAG3 mediated degradation could potentially have a different effect on SYNPO turnover. Thus, similar functions of SYNPO and SYNPO2 in protein degradation can not be excluded. Immunofluorescent staining of SYNPO in HeLa cells reveals a punctate pattern. These SYNPO punctae are largely increased in size upon overexpression of the largest of the three SYNPO isoforms: SYNPOc. These SYNPOc punctae often show small pointed protrusions and sometimes appear as ring like structures. Furthermore, it could be observed that BAG3 is recruited to these SYNPOc punctae. For elucidation of the molecular functions of SYNPO punctae and the role of SYNPO – BAG3 interaction, immunofluorescent costaining experiments with several marker proteins for e.g. aggresomes, the cytoskeleton, lysosomes or endosomes were conducted. A co-localization of SYNPOc punctae with the early endosome marker EEA1 (early endosome antigen 1) could be observed pointing to a potential involvement of SYNPO in endosomal transport or processing. In addition, new binding partners of SYNPO could be identified via immunoprecipitation followed by mass spectrometry, one of them being the actin associated protein Annexin A2 (ANXA2). These findings further underscore a potential involvement of SYNPO in endosomal transport, as ANXA2 has been previously described to play a role in endosomal trafficking. The exact mechanism of SYNPO involvement in endosomal transport, and how BAG3 might contribute to this process, remains to be a topic for further investigation. Besides confirming SYNPO as a novel binding partner of BAG3, this work provides fundamental information on SYNPO behavior in HeLa cells. The nature of BAG3 – SYNPO interaction could be further characterized and new interaction partners of SYNPO could be identified. In addition, this work provides first experimental data linking SYNPO, possibly together with BAG3, to endosomes, shedding light in potential molecular functions of SYNPO in HeLa cells

AHNAK and inflammatory markers predict poor survival in laryngeal carcinoma.

AHNAK/Desmoyokin is a giant protein which has been recently linked to reorganization of the actin cytoskeleton, cellular migration and invasion. Here, we investigated the role of AHNAK in the pathophysiology of larynx carcinoma-one of the major subtypes of head and neck cancer. To this end, we analysed AHNAK expression in tumor tissues from 83 larynx carcinoma patients in relation to overall survival. We found that tumoral AHNAK overexpression significantly associated with poor survival of these patients both in univariate and multivariate analysis. In further studies, we combined the prognostic value of AHNAK with selected markers of inflammation, such as macrophage migration inhibitory factor (MIF) and tumor-infiltrating neutrophils (CD66b-positive cells). Both MIF and neutrophils have been linked to enhanced tumoral migration and poor clinical outcome in patients with orohypopharynx carcinoma-another major subtype of head and neck cancer. Interestingly, we found that synchronous high levels of AHNAK and MIF or AHNAK and neutrophils, respectively, were stronger predictors of poor survival than AHNAK alone. Synchronous high levels of all three markers were the strongest predictors of poor survival in our patient cohort. Taken together, our findings propose novel strategies for an accurate prognosis in larynx carcinoma and suggest potential mechanisms of inflammation-mediated tumor progression

Neutrophils activate tumoral CORTACTIN to enhance progression of orohypopharynx carcinoma

CORTACTIN is an actin-binding protein critically involved in cellular migration and invasion. Here, we investigated the role of CORTACTIN in the pathophysiology of orohypopharynx carcinoma one of the major subtypes of head and neck cancer. To this end, we analyzed CORTACTIN expression in tumor tissues from 89 orohypopharynx carcinoma patients in relation to clinical parameters. We found that high tumoral CORTACTIN expression associated with poor survival, higher T-stage, and higher lymph node metastasis (N-stage) in these patients. Next, we combined the prognostic values of tumoral and stromal cell biological parameters in our patient cohort. We determined the potential interaction of tumoral CORTACTIN with tumor-infiltrating neutrophils, which have been previously linked to poor clinical outcome in orohypopharynx carcinoma patients with advanced disease. Interestingly, we found that patients with both high tumoral CORTACTIN expression and high neutrophilic infiltration had significantly worse clinical outcome than all other patients in our cohort. These findings suggest that tumoral CORTACTIN and tumor-infiltrating neutrophils might be functionally linked during progression of orohypopharynx carcinoma. In vitro, we showed that neutrophils released soluble factors which phosphorylated CORTACTIN in the tumor cells and promoted their migration. Furthermore, we demonstrated that strong CORTACTIN phosphorylation significantly correlated with strong neutrophilic infiltration in tumor tissues from orohypopharynx carcinoma patients. Taken together, our findings unravel a novel mechanism of tumor-stroma interaction, which might be relevant for a more accurate prognosis and improved therapeutic strategies in this tumor entity

A Prospective, Randomized, Double-blind, Vehicle-controlled, Multi-centre Clinical Trial of Efficacy, Safety and Local Tolerability

This study was a prospective, parallel-group, randomized, double-blind, vehicle-controlled, multi-centre clinical trial to compare the efficacy of topical sertaconazole 2% cream with vehicle in reducing chronic pruritus in subjects with atopic dermatitis, and to assess its safety and local tolerability. A total of 70 subjects applied either of the 2 treatments twice daily for a period of 4 weeks on affected, itchy skin areas. Treatment efficacy was evaluated primarily considering the item itch intensity on a 5-point verbal rating scale. Insomnia, state of atopic dermatitis (Scoring Atopic Dermatitis; SCORAD), quality of life and therapy benefit were also assessed. No significant difference between active treatment and vehicle was found at any of the time-points for any of the investigated parameters. Under the experimental conditions of the study, sertaconazole 2% cream did not exert anti-pruritic effects that were better than vehicle in subjects with atopic dermatitis who had chronic pruritus. Trial registration ClinicalTrials.gov #NCT01792713

Geschlechtsspezifische Unterschiede bei chronischem Pruritus

Chronischer Juckreiz ist mit einer Lebenszeitprävalenz von ca. 23 % ein sehr häufiges Symptom, das durch zahlreiche dermatologische, internistische, neurologische und auch psychische Erkrankungen ausgelöst werden kann. Während bei jüngeren Patientinnen und Patienten eher der entstellende Aspekt der durch Kratzen beschädigten Haut eine Rolle spielt, leiden ältere Patientinnen und Patienten oftmals unter einem schwer zu behandelbaren Juckreiz unterschiedlichster Ursache. Obwohl der chronische Pruritus als Volkssymptom angesehen werden kann, liegen bisher nur sehr wenige Studien zu geschlechtsspezifischen Unterschieden vor. Diese zeigen, dass Frauen und Männer eine unterschiedliche Pruritus- Wahrnehmung haben – Frauen nehmen das Symptom intensiver wahr. Dies führt bei Frauen nicht nur zu einer höheren psychischen Belastung, sondern auch zu einem unterschiedlichen Verhalten – Frauen kratzen vermehrt. Aber auch die Qualitäten des Symptoms sind unterschiedlich, Frauen empfinden beispielsweise vermehrt einen brennenden Juckreiz, was u. a. auf die Aktivierung von schmerzleitenden Nervenfasern (neuropathische Komponente) hindeutet. Dies deutet auf eine unterschiedliche Verarbeitung von Pruritus im Gehirn hin. Die geschlechtsspezifischen Unterschiede hinsichtlich der Juckempfindung sollten dringend weiter untersucht werden, um eine geschlechtsadaptierte Diagnostik und möglicherweise auch Therapie anbieten zu können und somit zur verbesserten Behandlung der Betroffenen beitragen zu können

Geschlechtsspezifische Unterschiede bei chronischem Pruritus

"Chronischer Juckreiz ist mit einer Lebenszeitprävalenz von ca. 23 % ein sehr häufiges Symptom, das durch zahlreiche dermatologische, internistische, neurologische und auch psychische Erkrankungen ausgelöst werden kann. Während bei jüngeren Patientinnen und Patienten eher der entstellende Aspekt der durch Kratzen beschädigten Haut eine Rolle spielt, leiden ältere Patientinnen und Patienten oftmals unter einem schwer zu behandelbaren Juckreiz unterschiedlichster Ursache. Obwohl der chronische Pruritus als Volkssymptom angesehen werden kann, liegen bisher nur sehr wenige Studien zu geschlechtsspezifischen Unterschieden vor. Diese zeigen, dass Frauen und Männer eine unterschiedliche Pruritus- Wahrnehmung haben - Frauen nehmen das Symptom intensiver wahr. Dies führt bei Frauen nicht nur zu einer höheren psychischen Belastung, sondern auch zu einem unterschiedlichen Verhalten - Frauen kratzen vermehrt. Aber auch die Qualitäten des Symptoms sind unterschiedlich, Frauen empfinden beispielsweise vermehrt einen brennenden Juckreiz, was u. a. auf die Aktivierung von schmerzleitenden Nervenfasern (neuropathische Komponente) hindeutet. Dies deutet auf eine unterschiedliche Verarbeitung von Pruritus im Gehirn hin. Die geschlechtsspezifischen Unterschiede hinsichtlich der Juckempfindung sollten dringend weiter untersucht werden, um eine geschlechtsadaptierte Diagnostik und möglicherweise auch Therapie anbieten zu können und somit zur verbesserten Behandlung der Betroffenen beitragen zu können." (Autorenreferat)"Chronicitch is a common symptom with a life-time prevalence of around 23% which is provoked by numerous dermatological, internal, neurological and mental disorders. While the disfiguring resulting from skin being damaged by scratching plays an important role in younger patients, older patients often suffer from a difficult-to-treat pruritus of various causes. Although chronic pruritus can be considered as a widespread disease, there are only a few studies which have examined sex-/ gender-specific differences. These studies have indicated that females and males have a different pruritus perception: females experience the symptom more intensively. This not only leads to a greater psychological burden in females, but results in a different behaviour, because females scratch more. Women also experience different symptoms, for example more women experience a burning itch, which indicates a stronger involvement of nerve fibres (neuropathic component). Women not only experience greater itch intensity, they are also more distracted by the itching than men are. This indicates a different cerebral perception and modulation. Sex-/genderspecific differences in regard to itching need further investigation in order to be able to offer sex-/gender-specific diagnostics and tailor therapy to improve the clinical situation of the affected patients." (Autorenreferat

Rapid prefractionation of complex protein lysates with centrifugal membrane adsorber units improves the resolving power of 2D-PAGE-based proteome analysis

BACKGROUND: Two-dimensional gel electrophoresis (2D-PAGE) has proven over the years to be a reliable and efficient method for separation of hundreds of proteins based on charge and mass. Nevertheless, the complexity of even the simplest proteomes limits the resolving power of 2D-PAGE. This limitation can be partially alleviated by sample prefractionation using a variety of techniques. RESULTS: Here, we have used Vivapure Ion Exchange centrifugal adsorber units to rapidly prefractionate total fission yeast protein lysate based on protein charge. Three fractions were prepared by stepwise elution with increasing sodium chloride concentrations. Each of the fractions, as well as the total lysate, were analyzed by 2D-PAGE. This simple prefractionation procedure considerably increased the resolving power of 2D-PAGE. Whereas 308 spots could be detected by analysing total protein lysate, 910 spots were observed upon prefractionation. Thorough gel image analysis demonstrated that prefractionation visualizes an additional set of 458 unique fission yeast proteins not detected in whole cell lysate. CONCLUSIONS: Prefractionation with Vivapure Q spin columns proved to be a simple, fast, reproducible, and cost-effective means of increasing the resolving power of 2D-PAGE using standard laboratory equipment

S2k guideline: Diagnosis and treatment of chronic pruritus

Pruritus is a cross-disciplinary leading symptom of numerous diseases and represents an interdisciplinary diagnostic and therapeutic challenge. In contrast to acute pruritus, chronic pruritus (CP) is a symptom of various diseases that is usually difficult to treat. Scratching and the development of scratch-associated skin lesions can alter the original skin status. In the presence of an itch-scratch-cycle, even secondary diseases such as chronic prurigo can develop. Chronic pruritus leads to considerable subjective suffering of those affected, which can result in restrictions on the health-related quality of life such as sleep disturbances, anxiety, depressiveness, experience of stigmatization and/or social withdrawal up to clinically relevant psychic comorbidities. Medical care of patients should therefore include (a) interdisciplinary diagnosis and therapy of the triggering underlying disease, (b) therapy of the secondary symptoms of pruritus (dermatological therapy, sleep promotion, in the case of an accompanying or underlying psychological or psychosomatic disease an appropriate psychological-psychotherapeutic treatment) and (c) symptomatic antipruritic therapy. The aim of this interdisciplinary guideline is to define and standardize the therapeutic procedure as well as the interdisciplinary diagnosis of CP. This is the short version of the updated S2k-guideline for chronic pruritus. The long version can be found at www.awmf.org

Scratching increases epidermal neuronal branching and alters psychophysical testing responses in atopic dermatitis and brachioradial pruritus

BackgroundChronic scratching imposes a major stress on the skin and can lead to itch intensity worsening, and consequently, patients may enter an itch–scratch cycle. This repetitive mechanical stress can result in lichenification, worsening of epidermal barrier function, and enhanced cutaneous inflammation. Furthermore, a reduction of intraepidermal nerve fibers was previously described in lichenification.AimThe aim of this study was to investigate the influence of chronic scratching on the epidermal neuroanatomy and on sensory changes, in particular the prevalence of hyperknesis and alloknesis in patients after mechanical, chemical, and electrical stimuli.MethodsAnalyses were performed on pruritic lichenified (chronically scratched), pruritic non-lichenified (not chronically scratched), and non-pruritic non-lesional (unaffected) skin areas of patients with inflammatory pruritus, i.e., atopic dermatitis (n = 35), and neuropathic pruritus, i.e., brachioradial pruritus (n = 34) vs. healthy matched controls (n = 64). Our fine-grained spatial skin characterization enabled specifically studying the differential effects of chronic scratching in inflammatory and neuropathic itch.ResultsAnalysis of intraepidermal nerve fiber density showed rarefaction of fibers in all three skin areas of patients compared with healthy controls in both diagnoses. Even more, the two pruritic areas had significantly less nerve fibers than the unaffected skin, whereas electrically induced itch was massively increased. Epidermal branching of the remaining nerve fibers in lichenified/chronically scratched skin was increased, particularly in patients with brachioradial pruritus, which may contribute to the pronounced local neuronal sensitivity. Hyperknesis and alloknesis were found to increase independently of lichenification.ConclusionOur results indicate that chronic scratching may not affect intraepidermal nerve fiber density but leads to a stronger branching pattern of intraepidermal nerve fibers, which may contribute to local hypersensitivity. The increased sensitivity in the pruritic areas suggests mechanisms of peripheral sensitization, whereas the increased sensation of electrically and chemically induced itch in unaffected skin indicates central sensitization for itch