Post-synaptic scaffold protein TANC2 in psychiatric and somatic disease risk

Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare TANC2 (Tetratricopeptide Repeat, Ankyrin Repeat and Coiled-Coil Containing 2) disrupting variants were disease-causing in NDD patients. This post-synaptic scaffold protein, essential for dendrite formation in synaptic plasticity, plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2 disrupted function model where mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.

Conformational heterogeneity coupled with β-fibril formation of a scaffold protein involved in chronic mental illnesses

Chronic mental illnesses (CMIs) pose a significant challenge to global health due to their complex and poorly understood etiologies and hence, absence of causal therapies. Research of the past two decades has revealed dysfunction of the disrupted in schizophrenia 1 (DISC1) protein as a predisposing factor involved in several psychiatric disorders. DISC1 is a multifaceted protein that serves myriads of functions in mammalian cells, for instance, influencing neuronal development and synapse maintenance. It serves as a scaffold hub forming complexes with a variety (~300) of partners that constitute its interactome. Herein, using combinations of structural and biophysical tools, we demonstrate that the C-region of the DISC1 protein is highly polymorphic, with important consequences for its physiological role. Results from solid-state NMR spectroscopy and electron microscopy indicate that the protein not only forms symmetric oligomers but also gives rise to fibrils closely resembling those found in certain established amyloid proteinopathies. Furthermore, its aggregation as studied by isothermal titration calorimetry (ITC) is an exergonic process, involving a negative enthalpy change that drives the formation of oligomeric (presumably tetrameric) species as well as β-fibrils. We have been able to narrow down the β-core region participating in fibrillization to residues 716–761 of full-length human DISC1. This region is absent in the DISC1Δ22aa splice variant, resulting in reduced association with proteins from the dynein motor complex, viz., NDE-like 1 (NDEL1) and lissencephaly 1 (LIS1), which are crucial during mitosis. By employing surface plasmon resonance, we show that the oligomeric DISC1 C-region has an increased affinity and shows cooperativity in binding to LIS1 and NDEL1, in contrast to the noncooperative binding mode exhibited by the monomeric version. Based on the derived structural models, we propose that the association between the binding partners involves two neighboring subunits of DISC1 C-region oligomers. Altogether, our findings highlight the significance of the DISC1 C-region as a crucial factor governing the balance between its physiological role as a multifunctional scaffold protein and aggregation-related aberrations with potential significance for disease.

Cost-Effective Production of ATP and S-Adenosylmethionine Using Engineered Multidomain Scaffold Proteins

Adenosine triphosphate (ATP) and S-adenosyl-L-methionine (SAM) are important intermediates that are widely present in living organisms. Large-scale preparation and application of ATP or SAM is limited by expensive raw materials. To lower the production costs for ATP/SAM, in this study we used strategies applying engineered multidomain scaffold proteins to synthesize ATP and SAM. An artificial scaffold protein containing CBM3 domain, IM proteins and CL-labeled proteins was assembled to form complex 1 for catalytic reactions to increase ATP production. The ATP synthesis system produced approximately 25 g/L of ATP with approximately 15 g/L of ADP and 5 g/L of AMP using 12.5 g/L of adenosine and 40 g/L of sodium hexametaphosphate reaction at 35 °C and a pH of 8.5 for 6 h. Based on the above ATP synthesis system, two CL-labeled methionine adenosyltransferases (CL9-MAT4 and CL9-MAT5) were applied to construct scaffold protein complex 2 to achieve SAM synthesis. Approximately 25 μg of MAT4 in a reaction system with 0.3 M MgCl2 catalyzed at 20 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 0.9 g/L of SAM. Approximately 25 μg of MAT5 in a reaction system with 0.7 M MgCl2 catalyzed at 35 °C and a pH of 8 catalyzed 0.5 g/L of l-Met to produce approximately 1.2 g/L of SAM. Here, we showed that low-cost substrates can be efficiently converted into high-value additional ATP and SAM via multi-enzyme catalytic reactions by engineered multidomain scaffold proteins.

Tks5 Regulates Synaptic Podosome Formation and Stabilization of the Postsynaptic Machinery at the Neuromuscular Junction

Currently, the etiology of many neuromuscular disorders remains unknown. Many of them are characterized by aberrations in the maturation of the neuromuscular junction (NMJ) postsynaptic machinery. Unfortunately, the molecular factors involved in this process are still largely unknown, which poses a great challenge for identifying potential therapeutic targets. Here, we identified Tks5 as a novel interactor of αdystrobrevin-1, which is a crucial component of the NMJ postsynaptic machinery. Tks5 has been previously shown in cancer cells to be an important regulator of actin-rich structures known as invadosomes. However, a role of this scaffold protein at a synapse has never been studied. We show that Tks5 is crucial for remodeling of the NMJ postsynaptic machinery by regulating the organization of structures similar to the invadosomes, known as synaptic podosomes. Additionally, it is involved in the maintenance of the integrity of acetylcholine receptor (AChR) clusters and regulation of their turnover. Lastly, our data indicate that these Tks5 functions may be mediated by its involvement in recruitment of actin filaments to the postsynaptic machinery. Collectively, we show for the first time that the Tks5 protein is involved in regulation of the postsynaptic machinery.

Drosophila Nesprin-1 Isoforms Differentially Contribute to Muscle Function

Nesprin-1 is a large scaffold protein connecting nuclei to the actin cytoskeleton via its KASH and Calponin Homology domains, respectively. Nesprin-1 disconnection from nuclei results in altered muscle function and myonuclei mispositioning. Furthermore, Nesprin-1 mutations are associated with muscular pathologies such as Emery Dreifuss muscular dystrophy and arthrogryposis. Nesprin-1 was thus proposed to mainly contribute to muscle function by controlling nuclei position. However, Nesprin-1′s localisation at sarcomere’s Z-discs, its involvement in organelles’ subcellular localization, as well as the description of numerous isoforms presenting different combinations of Calponin Homology (CH) and KASH domains, suggest that the contribution of Nesprin-1 to muscle functions is more complex. Here, we investigate the roles of Nesprin-1/Msp300 isoforms in muscle function and subcellular organisation using Drosophila larvae as a model. Subsets of Msp300 isoform were down-regulated by muscle-specific RNAi expression and muscle global function and morphology were assessed. We show that nuclei anchoring in mature muscle and global muscle function are disconnected functions associated with different Msp300 isoforms. Our work further uncovers a new and unsuspected role of Msp300 in myofibril registration and nuclei peripheral displacement supported by Msp300 CH containing isoforms, a function performed by Desmin in mammals.

Essential Drug Targets and Pathways in PI-Resistant MM Cells

Background Proteasome inhibitors (PI) have emerged as a powerful, cell biology-based treatment option for multiple myeloma (MM) and build a central backbone for MM treatment with three proteasome-inhibiting drugs currently approved: bortezomib (BTZ), carfilzomib (CFZ) and ixazomib. However, despite the high anti-MM activity of PI, MM cells adapt to the selective pressure of PI treatment in most cases to date and most MM patients relapse during or after treatment with PI, develop PI-refractory disease and ultimately die. Therefore, understanding and overcoming PI resistance is a key challenge for MM therapy. Our previous in vitro studies on PI-resistant MM suggest that PI-adapted, MM cells show very distinct features of general metabolism and cell biology that differentiate them from PI-sensitive MM, derived from the same cell line. We hypothesize that this highly specialized and adapted nature of PI-resistant MM offers novel areas of vulnerability, that differ from the therapeutic targets in PI-sensitive MM. The aim of our study was to identify essential drug targets and pathways in PI-resistant MM using genome-wide functional screening with the CRISPR/Cas9 system that could serve as novel therapeutic targets in PI-resistant MM. Methods We used genome-wide CRISPR/Cas9-based loss-of-function screening with Brunello library in L363-BTZ and RPMI-8226-BTZ cells, adapted to grow in the presence of 90 nM BTZ. The overlapping bortezomib genetic sensitivity candidates were further validated in the set of BTZ-resistant cells (L363-BTZ, RPMI-8226-BTZ, MM1S-BTZ and AMO-BTZ) cells using shRNA silencing or single-gene specific knockout or genetic overexpression using CCK8 viability assay. Subsequent functional analysis of the highest ranking BTZ sensitivity candidates in BTZ-adapted cells included apoptosis and cell cycle analysis, qPCR and western blotting, SILAC, proteasome activity determination using activity-based probes and FRAP analysis. Results CRISPR/Cas9-screening identified two candidate genes for BTZ sensitivity, ECPAS (KIAA0368; Ecm29 Proteasome Adaptor and Scaffold protein) and PSME1 (an 11S regulator complex subunit), as consistent screening hits in two independent BTZ-adapted MM cell lines. Both genes are related to proteasome, but do not build the proteasome core particle and do not have a proteolytic activity. Specific knock-down or knock-out of ECPAS sensitized PI-naïve cells to BTZ and CFZ, while significantly more sensitizing BTZ-adapted cells to both PI. Likewise, overexpression of PSMF1, an inhibitor of 11S regulator complex, sensitized BTZ-resistant as well as sensitive cells to BTZ. ECPAS-depleted BTZ-adapted cells showed accumulation of poly-ubiquitinated proteasome substrate proteins, induction of the unfolded protein response, cell cycle arrest and induction of apoptosis, together with changes in protein synthesis after the treatment with 50 nM bortezomib, in contrast to BTZ-adapted control cells. FRAP analysis of cells with GFP-tagged PSMD6 revealed that the intracellular mobility of proteasomes in ECPAS-depleted cells was reduced. Importantly, proteasome activity determined by activity-based probes was not impaired in ECPAS-depleted cells. Conclusion In conclusion, BTZ-resistant MM cells uniquely show a high dependency on the proteasome adaptor and scaffold protein ECPAS, which has been shown to be involved in coupling of proteasome in different compartments and promotes proteasome dissociation under oxidative stress. Specifically in PI-resistant MM, ECPAS is important to ensure functional proteasome, is involved in controlling the intracellular mobility of proteasomes, likely to ensure high proteasome turnover. ECPAS therefore represents a novel candidate that may be targeted to specifically re-sensitize PI-resistant MM cells to proteasome inhibitor treatment. Disclosures No relevant conflicts of interest to declare.

The GET pathway serves to activate Atg32-mediated mitophagy by ER targeting of the Ppg1-Far complex

Mitophagy removes defective or superfluous mitochondria via selective autophagy. In yeast, the pro-mitophagic protein Atg32 localizes to the mitochondrial surface and interacts with the scaffold protein Atg11 to promote degradation of mitochondria. Although Atg32-Atg11 interactions are thought to be stabilized by Atg32 phosphorylation, how this posttranslational modification is regulated remains obscure. Here we show that cells lacking the guided entry of tail-anchored proteins (GET) pathway exhibit reduced Atg32 phosphorylation and Atg32-Atg11 interactions, which can be rescued by additional loss of the ER-resident Ppg1-Far complex, a multi-subunit phosphatase negatively acting in mitophagy. In GET-deficient cells, Ppg1-Far is predominantly localized to mitochondria. An artificial ER anchoring of Ppg1-Far in GET-deficient cells significantly ameliorates defects in Atg32-Atg11 interactions and mitophagy. Moreover, disruption of GET and Msp1, an AAA-ATPase that extracts non-mitochondrial proteins localized to the mitochondrial surface, elicits synthetic defects in mitophagy. Collectively, we propose that the GET pathway mediates ER targeting of Ppg1-Far, thereby preventing dysregulated suppression of mitophagy activation.