Immuno- and constitutive proteasome crystal structures reveal differences in substrate and inhibitor specificity.

Constitutive proteasomes and immunoproteasomes shape the peptide repertoire presented by major histocompatibility complex class I (MHC-I) molecules by harboring different sets of catalytically active subunits. Here, we present the crystal structures of constitutive proteasomes and immunoproteasomes from mouse in the presence and absence of the epoxyketone inhibitor PR-957 (ONX 0914) at 2.9 Å resolution. Based on our X-ray data, we propose a unique catalytic feature for the immunoproteasome subunit ß5i/LMP7. Comparison of ligand-free and ligand-bound proteasomes reveals conformational changes in the S1 pocket of ß5c/X but not ß5i, thereby explaining the selectivity of PR-957 for ß5i. Time-resolved structures of yeast proteasome:PR-957 complexes indicate that ligand docking to the active site occurs only via the reactive head group and the P1 side chain. Together, our results support structure-guided design of inhibitory lead structures selective for immunoproteasomes that are linked to cytokine production and diseases like cancer and autoimmune disorders.

The Co-chaperone Cns1 and the Recruiter Protein Hgh1 Link Hsp90 to Translation Elongation via Chaperoning Elongation Factor 2.

The Hsp90 chaperone machinery in eukaryotes comprises a number of distinct accessory factors. Cns1 is one of the few essential co-chaperones in yeast, but its structure and function remained unknown. Here, we report the X-ray structure of the Cns1 fold and NMR studies on the partly disordered, essential segment of the protein. We demonstrate that Cns1 is important for maintaining translation elongation, specifically chaperoning the elongation factor eEF2. In this context, Cns1 interacts with the novel co-factor Hgh1 and forms a quaternary complex together with eEF2 and Hsp90. The in vivo folding and solubility of eEF2 depend on the presence of these proteins. Chaperoning of eEF2 by Cns1 is essential for yeast viability and requires a defined subset of the Hsp90 machinery as well as the identified eEF2 recruiting factor Hgh1.

Young hearts, early risks: novel cardiovascular biomarkers in former very preterm infants at kindergarten age.

BACKGROUND: Preterm birth is associated with long-term cardiovascular morbidity and mortality. In adults, fibroblast growth factor-23 (FGF-23), α-Klotho, and secretoneurin have all garnered attention as cardiovascular biomarkers, but their utility in pediatric populations has not yet been ascertained. The aim of this pilot study was to evaluate these novel cardiovascular biomarkers and their association with indicators of cardiovascular impairment in the highly vulnerable population of former very preterm infants. METHODS: Five- to seven-year-old children born at < 32 weeks' gestation were eligible for the study. Healthy same-aged children born at term served as controls. Biomarkers were quantified in fasting blood samples, and echocardiographic measurements including assessment of aortic elastic properties were obtained. RESULTS: We included 26 former very preterm infants and 21 term-born children in the study. At kindergarten age, former very preterm infants exhibited significantly higher plasma concentrations of biologically active intact FGF-23 (iFGF-23; mean 43.2 pg/mL vs. 29.1 pg/mL, p = 0.003) and secretoneurin (median 93.8 pmol/L vs. 70.5 pmol/L, p = 0.046). iFGF-23 inversely correlated with distensibility of the descending aorta. CONCLUSION: In preterm-born children, iFGF-23 and secretoneurin both offer prospects as valuable cardiovascular biomarkers, potentially allowing for risk stratification and timely implementation of preventive measures. IMPACT: Former very preterm infants have increased plasma concentrations of the novel cardiovascular biomarkers intact fibroblast growth factor-23 (iFGF-23) and secretoneurin at kindergarten age. Increases in iFGF-23 concentrations are associated with decreased distensibility of the descending aorta even at this early age. Monitoring of cardiovascular risk factors is essential in individuals with a history of preterm birth. Both iFGF-23 and secretoneurin hold promise as clinically valuable biomarkers for risk stratification, enabling the implementation of early preventive measures.

Epipolythiodioxopiperazine-Based Natural Products: Building Blocks, Biosynthesis and Biological Activities.

Epipolythiodioxopiperazines (ETPs) are fungal secondary metabolites that share a 2,5-diketopiperazine scaffold built from two amino acids and bridged by a sulfide moiety. Modifications of the core and the amino acid side chains, for example by methylations, acetylations, hydroxylations, prenylations, halogenations, cyclizations, and truncations create the structural diversity of ETPs and contribute to their biological activity. However, the key feature responsible for the bioactivities of ETPs is their sulfide moiety. Over the last years, combinations of genome mining, reverse genetics, metabolomics, biochemistry, and structural biology deciphered principles of ETP production. Sulfurization via glutathione and uncovering of the thiols followed by either oxidation or methylation crystallized as fundamental steps that impact expression of the biosynthesis cluster, toxicity and secretion of the metabolite as well as self-tolerance of the producer. This article showcases structure and activity of prototype ETPs such as gliotoxin and discusses the current knowledge on the biosynthesis routes of these exceptional natural products.

Association of growth with neurodevelopment in extremely low gestational age infants: a population-based analysis.

To assess the association between postnatal growth and neurodevelopment at the age of 2 years in extremely low gestational age newborns (ELGAN, < 28 weeks' gestation). Retrospective population-based cohort study including all live born ELGAN in 2006-2012 in Switzerland. Growth parameters (weight, length, head circumference, body mass index) were assessed at birth, at hospital discharge home, and 2-year follow-up (FU2). Unadjusted and adjusted regression models assessed associations between growth (birth to hospital discharge and birth to FU2) and neurodevelopment at FU2. A total of 1244 infants (mean GA 26.5 ± 1.0 weeks, birth weight 853 ± 189 g) survived to hospital discharge and were included in the analyses. FU2 was documented for 1049 (84.3%) infants. The mean (± SD) mental and a psychomotor development index at 2FU were 88.9 (± 18.0) and 86.9 (± 17.7), respectively. Moderate or severe neurodevelopmental impairment was documented in 23.2% of patients. Changes of z-scores between birth and discharge and between birth and FU2 for weight were - 1.06 (± 0.85) and - 0.140 (± 1.15), for length - 1.36 (± 1.34), and - 0.40 (± 1.33), for head circumference - 0.61 (± 1.04) and - 0.76 (± 1.32) as well as for BMI 0.22 (± 3.36) and - 0.006 (± 1.45). Unadjusted and adjusted analyses showed that none of the four growth parameters was significantly associated with any of the three outcome parameters of neurodevelopment. This was consistent for both time intervals. CONCLUSION: In the present population-based cohort of ELGAN, neither growth between birth and hospital discharge nor between birth and FU2 were significantly associated with neurodevelopment at age of 2 years. WHAT IS KNOWN: • Studies assessing the association between growth and neurodevelopment in extremely low gestational age newborns (28 weeks' gestation) show conflicting results. WHAT IS NEW: • Neither growth between birth and hospital discharge nor between birth and corrected age of 2 years were significantly associated with neurodevelopment at age of 2 years. • The role of postnatal growth as a predictor of neurodevelopmental outcome during infancy might be smaller than previously assumed.

Structural and Mechanistic Insights into C-S Bond Formation in Gliotoxin.

Glutathione-S-transferases (GSTs) usually detoxify xenobiotics. The human pathogenic fungus Aspergillus fumigatus however uses the exceptional GST GliG to incorporate two sulfur atoms into its virulence factor gliotoxin. Because these sulfurs are essential for biological activity, glutathionylation is a key step of gliotoxin biosynthesis. Yet, the mechanism of carbon-sulfur linkage formation from a bis-hydroxylated precursor is unresolved. Here, we report structures of GliG with glutathione (GSH) and its reaction product cyclo[-l-Phe-l-Ser]-bis-glutathione, which has been purified from a genetically modified A. fumigatus strain. The structures argue for stepwise processing of first the Phe and second the Ser moiety. Enzyme-mediated dehydration of the substrate activates GSH and a helix dipole stabilizes the resulting anion via a water molecule for the nucleophilic attack. Activity assays with mutants validate the interactions of GliG with the ligands and enrich our knowledge about enzymatic C-S bond formation in gliotoxin and epipolythiodioxopiperazine (ETP) natural compounds in general.

A humanized yeast proteasome identifies unique binding modes of inhibitors for the immunosubunit ß5i.

Inhibition of the immunoproteasome subunit ß5i alleviates autoimmune diseases in preclinical studies and represents a promising new anti-inflammatory therapy. However, the lack of structural data on the human immunoproteasome still hampers drug design. Here, we systematically determined the potency of seven α' ß' epoxyketone inhibitors with varying N-caps and P3-stereochemistry for mouse/human ß5c/ß5i and found pronounced differences in their subunit and species selectivity. Using X-ray crystallography, the compounds were analyzed for their modes of binding to chimeric yeast proteasomes that incorporate key parts of human ß5c, human ß5i or mouse ß5i and the neighboring ß6 subunit. The structural data reveal exceptional conformations for the most selective human ß5i inhibitors and highlight subtle structural differences as the major reason for the observed species selectivity. Altogether, the presented results validate the humanized yeast proteasome as a powerful tool for structure-based development of ß5i inhibitors with potential clinical applications.

Maternal high-fat diet induces long-term obesity with sex-dependent metabolic programming of adipocyte differentiation, hypertrophy and dysfunction in the offspring.

Maternal obesity determines obesity and metabolic diseases in the offspring. The white adipose tissue (WAT) orchestrates metabolic pathways, and its dysfunction contributes to metabolic disorders in a sex-dependent manner. Here, we tested if sex differences influence the molecular mechanisms of metabolic programming of WAT in offspring of obese dams. To this end, maternal obesity was induced with high-fat diet (HFD) and the offspring were studied at an early phase [postnatal day 21 (P21)], a late phase (P70) and finally P120. In the early phase we found a sex-independent increase in WAT in offspring of obese dams using magnetic resonance imaging (MRI), which was more pronounced in females than males. While the adipocyte size increased in both sexes, the distribution of WAT differed in males and females. As mechanistic hints, we identified an inflammatory response in females and a senescence-associated reduction in the preadipocyte factor DLK in males. In the late phase, the obese body composition persisted in both sexes, with a partial reversal in females. Moreover, female offspring recovered completely from both the adipocyte hypertrophy and the inflammatory response. These findings were linked to a dysregulation of lipolytic, adipogenic and stemness-related markers as well as AMPKα and Akt signaling. Finally, the sex-dependent metabolic programming persisted with sex-specific differences in adipocyte size until P120. In conclusion, we do not only provide new insights into the molecular mechanisms of sex-dependent metabolic programming of WAT dysfunction, but also highlight the sex-dependent development of low- and high-grade pathogenic obesity.

Responding to self-criticism in psychotherapy.

OBJECTIVE: We explored the interactive process in which therapists respond to client self-critical positions. METHODS: Drawing from the resources of conversation analysis (CA), we examined a corpus of in-session self-critical sequences of talk occurring in different kinds of treatments: Client Centered Therapy, (CCT), Emotion Focused Therapy (EFT), Psychoanalytic Psychotherapy (PP) and in different cultural contexts. RESULTS: It was found that client self-critical talk performed various functions pertaining to diminished control, accountability (e.g., failed obligations leading to self-blame) and disparaging evaluations of self (contempt or disgust). Further, therapists were found to respond in ways that targeted the client's report of having diminished control or of being accountable for their negative attributes by providing a more optimistic reading of the client's experience, one that is more open to positive outcomes and the possibility of change. Our sequential analysis not only shows how clients may resist these optimistic readings, but also how therapists work towards successfully achieving moments of re-affiliation. CONCLUSION: We anticipate that the fine-grained sequential analysis of therapy interaction can provide therapists with a more detailed understanding of the options and challenges therapists face when working with clinical challenges of clients' self-critical positions.

[Levels of structural integration in adolescents and the relationship to later mental disorders - A longitudinal study]. / Psychisches Strukturniveau im Jugendalter und der Zusammenhang mit späterer psychischer Erkrankung ­ eine Langzeitstudie.

Levels of structural integration in adolescents and the relationship to later mental disorders - A longitudinal study Abstract. Objective: Psychological disorders frequently manifest during adolescence. Because of the multifactorial influencing factors, the courses of the diseases are heterogeneous, from relapsing-remitting to chronic. This study investigated whether the level of structural integration of the Operationalized Psychodynamic Diagnostics in Childhood and Adolescence (OPD-CA) correlates with later symptomatic burden. Method: This long-term study assessed the levels of structural integration according to the OPD-CA of 60 adolescents (mean age = 15.6; SD = 0.9). Seven years later, we then measured symptomatic burden (SCID axis I and II) and overall burden (GAF, BSI-GSI) (73.3 % follow-up participation rate). Results: The results showed high correlations between deficient structural integration in adolescence and later symptoms and overall burden in early adulthood. Conclusion: The follow-up examination after a 7-year time period showed significant correlations, which argue for the predictive value of structural integration. This suggests that early specific treatment, e.g., in the form of intensive psychotherapy, be urgently recommended in order to influence this course.

[Operationalized Psychodynamic Diagnosis - Structure Questionnaire (OPD-SQ) in Youth: First Results on Reliability and Validity]. / Der OPD-Strukturfragebogen (OPD-SF) in Anwendung auf eine jugendliche Schul- und klinische Stichprobe: Ergebnisse zu Reliabilität und Validität.

Operationalized Psychodynamic Diagnosis - Structure Questionnaire (OPD-SQ) in Youth: First Results on Reliability and Validity The OPD structure axis questionnaire (OPD-SQ) demonstrated good empirical findings and usability in therapeutic work. Therefore, the aim of this study was to verify the OPD-SQ for an adolescent sample in order to further implement the questionnaire as an economic instrument in clinical childhood and adolescence psychiatry routine. The overall sample consisted of 180 adolescent inpatients (72.8 % female) and 152 pupils (57.9 % female). In order to test reliability, Cronbach's alpha and discriminatory power were assessed. To test validity, YSR11-18 data was collected for the overall sample, and further data (AIDA, LoPF Q12-18, SKID-II, WISC-IV) was collected for the inpatient-sample. Cronbach's alpha was satisfactory for overall OPD-SQ, and all dimensions and sub-dimensions. The differences in mean values between inpatients and pupils were as expected. When controlled for overall symptom severity, gender differences lost significance. Adolescents with one or more SCID-II diagnoses had significantly lower psychic structure. Both construct-related questionnaires (AIDA, LoPF Q12-18) correlated high with OPD-SQ, no correlations were found for psychic structure and intelligence. OPD-SQ proved to be well applicable for the use on adolescents and can be implemented as an economic instrument for the measure of psychic structure in clinical adolescent psychiatry routine.

Iron Scavenging in Aspergillus Species: Structural and Biochemical Insights into Fungal Siderophore Esterases.

Fungi utilize high-affinity chelators termed siderophores with chemically diverse structures to scavenge the essential nutrient iron from their surroundings. Since they are among the strongest known Fe3+ binding agents, intracellular release of the heavy metal atom is facilitated by the activity of specific hydrolases. In this work, we report the characterization and X-ray crystal structures of four siderophore esterases: AfEstB and AfSidJ from Aspergillus fumigatus, as well as AnEstB and AnEstA from Aspergillus nidulans. Even though they all display the conserved α/ß-hydrolase fold, we found significant structural and enzymatic discrepancies in their adaption to both related and chemically diverse substrates. A structure of AfEstB in complex with its substrate triacetylfusarinine C gives insight into the active enzyme and shows tetrahedral coordination between the catalytic serine and the scissile ester bond.

Structural Elucidation of a Nonpeptidic Inhibitor Specific for the Human Immunoproteasome.

Selective inhibition of the immunoproteasome is a promising approach towards the development of immunomodulatory drugs. Recently, a class of substituted thiazole compounds that combine a nonpeptidic scaffold with the absence of an electrophile was reported in a patent. Here, we investigated the mode of action of the lead compound by using a sophisticated chimeric yeast model of the human immunoproteasome for structural studies. The inhibitor adopts a unique orientation perpendicular to the ß5i substrate-binding channel. Distinct interactions between the inhibitor and the subpockets of the human immunoproteasome account for its isotype selectivity.

Levels of Structural Integration and Facial Expressions of Negative Emotions.

OBJECTIVES: For a clinically relevant understanding of facial displays of patients with mental disorders it is crucial to go beyond merely counting frequencies of facial expressions, but include the contextual information of the expression. We assume that patients with different levels of structural integration differ in the contextual embedding of their negative facial expressions of emotions. METHODS: Facial affective behaviour of 80 female participants during an OPD interview was analysed using FACS (Facial Action Coding System) and the RFE coding system (Referencesof- Facial-Expression coding system; Bock et al. 2015).Using the RFE coding system, 2192 negative facial expressions of emotions were attributed to different references (e.g., interactive, self-related, object-related) by relying on contextual variables. RESULTS: Pure frequency of negative facial affect was not related to level of structural integration. Negative facial expressions of emotions directed towards the interviewer (interactive reference), as well as negative facial expressions directed towards the displayer's whole self were associated with lower levels of structural integration. In contrast, negative facial affects directed to single aspects of the self, to single aspects of objects, or to external situations were associated with higher levels of structural integration. CONCLUSIONS: The differentiation of references of facial affective behavior allows a deeper understanding of the connection between facial displays and structural levels of psychic integration.

Systematic Analyses of Substrate Preferences of 20S Proteasomes Using Peptidic Epoxyketone Inhibitors.

Cleavage analyses of 20S proteasomes with natural or synthetic substrates allowed to infer the substrate specificities of the active sites and paved the way for the rational design of high-affinity proteasome inhibitors. However, details of cleavage preferences remained enigmatic due to the lack of appropriate structural data. In a unique approach, we here systematically examined substrate specificities of yeast and human proteasomes using irreversibly acting α',ß'epoxyketone (ep) inhibitors. Biochemical and structural analyses provide unique insights into the substrate preferences of the distinct active sites and highlight differences between proteasome types that may be considered in future inhibitor design efforts. (1) For steric reasons, epoxyketones with Val or Ile at the P1 position are weak inhibitors of all active sites. (2) Identification of the ß2c selective compound Ac-LAE-ep represents a promising starting point for the development of compounds that discriminate between ß2c and ß2i. (3) The compound Ac-LAA-ep was found to favor subunit ß5c over ß5i by three orders of magnitude. (4) Yeast ß1 and human ß1c subunits preferentially bind Asp and Leu in their S1 pockets, while Glu and large hydrophobic residues are not accepted. (5) Exceptional structural features in the ß1/2 substrate binding channel give rise to the ß1 selectivity of compounds featuring Pro at the P3 site. Altogether, 23 different epoxyketone inhibitors, five proteasome mutants, and 43 crystal structures served to delineate a detailed picture of the substrate and ligand specificities of proteasomes and will further guide drug development efforts toward subunit-specific proteasome inhibitors for applications as diverse as cancer and autoimmune disorders.

[Facial expressions of negative emotions in clinical interviews: The development, reliability and validity of a categorical system for the attribution of functions to facial expressions of negative emotions]. / Negative mimische Affekte im Kontext klinischer Interviews: Entwicklung, Reliabilität und Validität einer Methode zur Funktionsbestimmung negativer Affektmimik.

OBJECTIVES: The development (Study 1) and validation (Study 2) of a categorical system for the attribution of facial expressions of negative emotions to specific functions. METHOD: The facial expressions observed inOPDinterviews (OPD-Task-Force 2009) are coded according to the Facial Action Coding System (FACS; Ekman et al. 2002) and attributed to categories of basic emotional displays using EmFACS (Friesen & Ekman 1984). In Study 1 we analyze a partial sample of 20 interviews and postulate 10 categories of functions that can be arranged into three main categories (interactive, self and object). In Study 2 we rate the facial expressions (n=2320) from the OPD interviews (10 minutes each interview) of 80 female subjects (16 healthy, 64 with DSM-IV diagnosis; age: 18-57 years) according to the categorical system and correlate them with problematic relationship experiences (measured with IIP,Horowitz et al. 2000). RESULTS: Functions of negative facial expressions can be attributed reliably and validly with the RFE-Coding System. CONCLUSIONS: The attribution of interactive, self-related and object-related functions allows for a deeper understanding of the emotional facial expressions of patients with mental disorders.

A Minimal ß-Lactone Fragment for Selective ß5c or ß5i Proteasome Inhibitors.

Broad-spectrum proteasome inhibitors are applied as anticancer drugs, whereas selective blockage of the immunoproteasome represents a promising therapeutic rationale for autoimmune diseases. We here aimed at identifying minimal structural elements that confer ß5c or ß5i selectivity on proteasome inhibitors. Based on the natural product belactosin C, we synthesized two ß-lactones featuring a dimethoxybenzyl moiety and either a methylpropyl (pseudo-isoleucin) or an isopropyl (pseudo-valine) P1 side chain. Although the two compounds differ only by one methyl group, the isoleucine analogue is six times more potent for ß5i (IC50=14 nM) than the valine counterpart. Cell culture experiments demonstrate the cell-permeability of the compounds and X-ray crystallography data highlight them as minimal fragments that occupy primed and non-primed pockets of the active sites of the proteasome. Together, these results qualify ß-lactones as a promising lead-structure motif for potent nonpeptidic proteasome inhibitors with diverse pharmaceutical applications.

Selective Inhibition of the Immunoproteasome by Structure-Based Targeting of a Non-catalytic Cysteine.

Clinically applied proteasome inhibitors induce cell death by concomitant blockage of constitutive and immunoproteasomes. In contrast, selective immunoproteasome inhibition is less cytotoxic and has the potential to modulate chronic inflammation and autoimmune diseases. In this study, we rationally designed decarboxylated peptides that covalently target a non-catalytic cysteine of the immunoproteasome subunit ß5i with α-chloroacetamide-containing sidechains. The enhanced isoform specificity decreased cytotoxic effects and the compound suppressed the production of inflammatory cytokines. Structure-based optimization led to over 150-fold selectivity for subunit ß5i over ß5c. This new compound class provides a promising starting point for the development of selective immunoproteasome inhibitors as potential anti-inflammatory agents.

Flavoenzyme-catalyzed formation of disulfide bonds in natural products.

Nature provides a rich source of compounds with diverse chemical structures and biological activities, among them, sulfur-containing metabolites from bacteria and fungi. Some of these compounds bear a disulfide moiety that is indispensable for their bioactivity. Specialized oxidoreductases such as GliT, HlmI, and DepH catalyze the formation of this disulfide bridge in the virulence factor gliotoxin, the antibiotic holomycin, and the anticancer drug romidepsin, respectively. We have examined all three enzymes by X-ray crystallography and activity assays. Despite their differently sized substrate binding clefts and hence, their diverse substrate preferences, a unifying reaction mechanism is proposed based on the obtained crystal structures and further supported by mutagenesis experiments.