Chaperone domains convert prolyl isomerases into generic catalysts of protein folding

The cis/trans isomerization of peptide bonds before proline (prolyl bonds) is a rate-limiting step in many protein folding reactions, and it is used to switch between alternate functional states of folded proteins. Several prolyl isomerases of the FK506-binding protein family, such as trigger factor, SlyD, and FkpA, contain chaperone domains and are assumed to assist protein folding in vivo. The prolyl isomerase activity of FK506-binding proteins strongly depends on the nature of residue Xaa of the Xaa-Pro bond. We confirmed this in assays with a library of tetrapeptides in which position Xaa was occupied by all 20 aa. A high sequence specificity seems inconsistent with a generic function of prolyl isomerases in protein folding. Accordingly, we constructed a library of protein variants with all 20 aa at position Xaa before a rate-limiting cis proline and used it to investigate the performance of trigger factor and SlyD as catalysts of proline-limited folding. The efficiencies of both prolyl isomerases were higher than in the tetrapeptide assays, and, intriguingly, this high activity was almost independent of the nature of the residue before the proline. Apparently, the almost indiscriminate binding of the chaperone domain to the refolding protein chain overrides the inherently high sequence specificity of the prolyl isomerase site. The catalytic performance of these folding enzymes is thus determined by generic substrate recognition at the chaperone domain and efficient transfer to the active site in the prolyl isomerase domain.     

Deformation strain is the main physical driver for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation

Mesenchymal stem cells play a major role during bone remodelling and are thus of high interest for tissue engineering and regenerative medicine applications. Mechanical stimuli, that is, deformation strain and interstitial fluid‐flow‐induced shear stress, promote osteogenic lineage commitment. However, the predominant physical stimulus that drives early osteogenic cell maturation is not clearly identified. The evaluation of each stimulus is challenging, as deformation and fluid‐flow‐induced shear stress interdepend. In this study, we developed a bioreactor that was used to culture mesenchymal stem cells harbouring a strain‐responsive AP‐1 luciferase reporter construct, on porous scaffolds. In addition to the reporter, mineralization and vitality of the cells was investigated by alizarin red staining and 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide. Quantification of the expression of genes associated to bone regeneration and bone remodelling was used to confirm alizarin red measurements. Controlled perfusion and deformation of the 3‐dimensional scaffold facilitated the alteration of the expression of osteogenic markers, luciferase activity, and calcification. To isolate the specific impact of scaffold deformation, a computational model was developed to derive a perfusion flow profile that results in dynamic shear stress conditions present in periodically loaded scaffolds. In comparison to actually deformed scaffolds, a lower expression of all measured readout parameters indicated that deformation strain is the predominant stimulus for skeletal precursors to undergo osteogenesis in earlier stages of osteogenic cell maturation.     

Obsessive–Compulsive Disorder is Characterized by a Lack of Adaptive Coping Rather than an Excess of Maladaptive Coping

The present study aimed to elucidate the profile of coping in patients with obsessive–compulsive disorder (OCD) in order to discern whether the disorder is characterized by an excess of maladaptive coping skills and/or a lack of adaptive coping skills. Sixty individuals with OCD were compared with 110 individuals with depression and 1050 nonclinical controls on the Maladaptive and Adaptive Coping Styles Questionnaire (MAX). Psychopathology was assessed with the Obsessive–Compulsive Inventory-Revised (OCI-R), the Yale-Brown Obsessive–Compulsive Scale (Y-BOCS), and the Patient Health Questionnaire-9 for depression (PHQ-9). Individuals with OCD and depression displayed more maladaptive coping and avoidance as well as less adaptive coping than nonclinical controls. Importantly, adaptive coping was significantly lower in individuals with OCD than in those with depression at a medium effect size, whereas the clinical groups were indistinguishable on maladaptive coping and avoidance. Lack of adaptive coping was strongly correlated with resistance to symptoms and poor insight in OCD (Y-BOCS), even after controlling for depression. Lack of adaptive coping skills may represent a specific pathogenetic factor in OCD. Longitudinal studies need to clarify whether strengthening adaptive skills during childhood and adolescence may help to prevent the progression from subclinical to manifest OCD.     

Synergistic interaction of the histone deacetylase inhibitor SAHA with the proteasome inhibitor bortezomib in mantle cell lymphoma

Objectives:  Mantle cell lymphoma (MCL) is an incurable B cell lymphoma, and novel treatment strategies are urgently needed. We evaluated the effects of combined treatment with the proteasome inhibitor bortezomib and the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) on MCL. Bortezomib acts by targeting the proteasome, and – among other mechanisms – results in a reduced nuclear factor-kappa B (NF-κB) activity. HDACi promote histone acetylation, and also interfere with NF-κB signaling.
Methods:  Human MCL cell lines (JeKo-1, Granta-519 and Hbl-2) were exposed to bortezomib and/or SAHA. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. Reactive oxygen species (ROS) were analyzed using the fluorophore H₂DCFDA. In addition, activated caspases, proteasome- and NF-κB activity were quantified.
Results:  Combined incubation with bortezomib and SAHA resulted in synergistic cytotoxic effects, as indicated by combination index values <1 using the median effect method of Chou and Talalay. The combination of both inhibitors led to a strong increase in apoptosis as compared to single agents and was accompanied by enhanced ROS generation, while each agent alone only modestly induced ROS. The free radical scavenger N -acetyl- l -cysteine blocked the ROS generation and reduced the apoptosis significantly. In addition, coexposure of bortezomib and SAHA led to increased caspase-3, -8 and -9 activity, marked reduction of proteasome activity and decrease of NF-κB activity.
Conclusions:  This is the first report giving evidence that SAHA and bortezomib synergistically induce apoptosis in MCL cells. These data build the framework for clinical trials using combined proteasome and histone deacetylase inhibition in the treatment of MCL.