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The functionality of most secreted proteins depends on their assembly into a defined quaternary structure. Despite this, it remains unclear how cells discriminate unassembled proteins en route to the native state from misfolded ones that need to be degraded. Here we show how chaperones can regulate and control assembly of heterodimeric proteins, using interleukin 23 (IL-23) as a model. We find that the IL-23 α-subunit remains partially unstructured until assembly with its β-subunit occurs and identify a major site of incomplete folding. Incomplete folding is recognized by different chaperones along the secretory pathway, realizing reliable assembly control by sequential checkpoints. Structural optimization of the chaperone recognition site allows it to bypass quality control checkpoints and provides a secretion-competent IL-23α subunit, which can still form functional heterodimeric IL-23. Thus, locally-restricted incomplete folding within single-domain proteins can be used to regulate and control their assembly., It is unclear how unassembled secretory pathway proteins are discriminated from misfolded ones. Here the authors combine biophysical and cellular experiments to study the folding of heterodimeric interleukin 23 and describe how ER chaperones recognize unassembled proteins and aid their assembly into protein complexes while preventing the premature degradation of unassembled units.

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V-L) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the VL and CL domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of VL and CL to each other are of major importance for maintaining the structural integrity of the full-length LC. (C) 2018 Elsevier Ltd. All rights reserved.

Constitutive activation of the antiapoptotic nuclear factor-κB (NF-κB) signaling pathway is a hallmark of the activated B-cell-like (ABC) subtype of diffuse large B-cell lymphomas (DLBCL). Recurrent oncogenic mutations are found in the scaffold protein CARMA1 (CARD11) that connects B-cell receptor (BCR) signaling to the canonical NF-κB pathway. We asked how far additional downstream processes are activated and contribute to the oncogenic potential of DLBCL-derived CARMA1 mutants. To this end, we expressed oncogenic CARMA1 in the NF-κB negative DLBCL lymphoma cell line BJAB. By a proteomic approach we identified recruitment of β-catenin and its destruction complex consisting of APC, AXIN1, CK1α and GSK3β to oncogenic CARMA1. Recruitment of the β-catenin destruction complex was independent of CARMA1-BCL10-MALT1 complex formation or constitutive NF-κB activation and promoted the stabilization of β-catenin. The β-catenin destruction complex was also recruited to CARMA1 in ABC DLBCL cell lines, which coincided with elevated β-catenin expression. In line, β-catenin was frequently detected in non-GCB DLBCL biopsies that rely on chronic BCR signaling. Increased β-catenin amounts alone were not sufficient to induce classical WNT target gene signatures, but could augment TCF/LEF-dependent transcriptional activation in response to WNT signaling. In conjunction with NF-κB, β-catenin enhanced expression of immunosuppressive interleukin-10 and suppressed antitumoral CCL3, indicating that β-catenin can induce a favorable tumor microenvironment. Thus, parallel activation of NF-κB and β-catenin signaling by gain-of-function mutations in CARMA1 augments WNT stimulation and is required for regulating the expression of distinct NF-κB target genes to trigger cell-intrinsic and extrinsic processes that promote DLBCL lymphomagenesis.

Peroxisome proliferator-activated receptor β (PPARβ) is a ligand-induced transcription factor. PPARβ plays essential roles in lipid metabolism and energy homoeostasis and has important functions in the regulation of cell proliferation, differentiation and apoptosis. Additionally, previous work of our group unravelled an unexpected role in tumour angiogenesis. The genetic disruption of the Pparb gene leads to tumour endothelial hyperplasia and deregulation of tumour angiogenesis. As a consequence syngeneic lewis lung tumour growth is impaired in Pparb-deficient mice. In the present thesis it could be shown that the retroviral transduction of PPARβ triggers microvessel differentiation and maturation in an in vivo model system (matrigel). Several potential PPARβ target genes were previously identified in the same model. As part of the present work, the PPARβ responsiveness of these target genes was validated and characterised in various cell culture systems (genetic disruption, reexpression and RNAi knockdown of Pparb). In addition to the known regulators of angiogenesis Cd36 and Thbs2, we identified the Cdkn1c gene encoding the cell cycle inhibitor p57KIP2 as a PPARβ target gene. Furthermore, a functional connection between PPARβ and p57KIP2 in the regulation of fibroblast proliferation could be demonstrated. In the second part of this work, the influence of ligands and intracellular fatty acid transport proteins on the transcriptional activity of the PPARβ receptor has been analysed. All trans-retinoic acid (atRA) has recently been reported to act as a ligand for PPARβ, to activate its transcriptional activity and, in contrast to the “classical” function of atRA, to stimulate cell proliferation. In contrast to these findings, results of the present work rule out a direct activation of PPARβ and its target genes by atRA. Additionally, no influence of the fatty acid binding protein 5 (FABP5) on transcriptional activity of PPARβ could be detected. The third part of this work addressed the question as to whether PPARβ is post-translational modified by ubiquitin or SUMO, as previously reported for PPARα and / or PPARγ. In the present thesis, SUMOylation of PPARβ was undetectable, whereas modification by ubiquitination and degradation by the 26S-proteasome system could be clearly demonstrated. Additionally, a stabilising influence of ligand-binding was seen only under conditions of PPARβ overexpression, which might serve as a safeguard mechanism protecting the cell from deregulated PPARβ expression under certain pathophysiological conditions., Der Peroxisomen-Proliferator aktivierte Rezeptor β (PPARβ) ist ein Liganden-induzierter Transkriptionsfaktor. PPARβ spielt eine wichtige Rolle im Lipidmetabolismus und bei der Energiehomöostase, weiterhin übernimmt er eine regulatorische Funktion bei der Zelldifferenzierung, Proliferation und Apoptose. Zudem zeigten Vorarbeiten unserer Arbeitsgruppe eine Funktion in der Tumorangiogenese. Demnach führt die genetische Inaktivierung des Rezeptors zu einem hyperplastischen Phänotyp der in den Tumor einwachsenden Endothelzellen (ECs). Die Folge ist ein Vaskularisierungsdefekt, der für die Inhibition des Wachstums von syngenen Lewis Lung Tumoren in der Maus verantwortlich ist. In der vorliegenden Arbeit konnte gezeigt werden, dass die retrovirale Reexpression von PPARβ zu einer verstärkten Reifung und Differenzierung von Endothelzellen in einem in vivo Modellsystem (Matrigel) führt. Im gleichen Modell waren bereits potentielle Zielgene von PPARβ identifiziert worden, die im Rahmen dieser Arbeit in verschiedenen experimentellen Systemen in Zellkultur validiert und charakterisiert wurden (genetische Inaktivierung, Reexpression und RNAi-vermittelte Inhibition von Pparb). Dabei konnte neben den Genen der bekannten Angiogenese-Regulatoren Cd36 und Thbs2 auch das für den Zellzyklusinhibitor p57KIP2 codierende Cdkn1c Gen als positiv reguliertes PPARβ Zielgen nachgewiesen werden. Zudem konnte ein funktionaler Zusammenhang zwischen PPARβ und p57KIP2 bei der Regulation der Proliferation von Fibroblasten gezeigt werden. Im zweiten Teil dieser Arbeit sollte der Einfluss von Liganden und Transportproteinen auf die transkriptionelle Aktivität des Rezeptors analysiert werden. In einer kürzlich erschienenen Publikation wurde all-trans Retinsäure (atRA) als PPARβ Ligand beschrieben und sollte, im Gegensatz zu seiner bekannten Funktion als Ligand für die Retinsäure-Rezeptoren, über eine Bindung an PPARβ die Zellproliferation stimulieren. Untersuchungen der vorliegenden Dissertation konnten eine transkriptionelle Aktivierung von PPARβ und seiner Zielgene durch atRA sowie einen Einfluss des Fettsäuretransporters FABP5 jedoch eindeutig widerlegen. Die Fragestellung des dritten Teils dieser Arbeit war die nach einer möglichen posttranslationalen Modifikation von PPARβ durch Ubiquitinierung oder SUMOylierung, wie sie teilweise auch schon für PPARα und PPARγ beschrieben wurde. In der vorliegenden Arbeit konnte zwar keine SUMOylierung von PPARβ nachgewiesen werden, jedoch eine deutliche Modifikation durch Ubiquitin und Degradation über das 26S-Proteasomsystem. Ein stabilisierender Einfluss von Liganden auf die Halbwertszeit von PPARβ wurde spezifisch nach dessen Überexpression beobachtet und könnte somit unter bestimmten pathophysiologischen Bedingungen relevant sein.