SUMMARY The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a “normal” human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor micro-tubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening.

Summary Degradation by the proteasome typically requires substrate ubiquitination. Two ubiquitin receptors exist in the proteasome, S5a/Rpn10 and Rpn13. Whereas Rpn13 has only one ubiquitin-binding surface, S5a binds ubiquitin with two independent ubiquitin interacting motifs (UIMs). Here, we use NMR and analytical ultracentrifugation to define at atomic level resolution how S5a binds K48-linked diubiquitin, in which K48 of one ubiquitin subunit (the “proximal” one) is covalently bonded to G76 of the other (the “distal” subunit). We demonstrate that S5a’s UIMs bind the two subunits simultaneously with a preference for UIM2 binding to the proximal subunit while UIM1 binds to the distal one. In addition, NMR experiments reveal that Rpn13 and S5a bind K48-linked diubiquitin simultaneously with subunit specificity, and a model structure of S5a and Rpn13 bound to K48-linked polyubiquitin is provided. Altogether, our data demonstrate that S5a is highly adaptive and cooperative towards binding ubiquitin chains.

The serum response factor (SRF) binds to coactivators, such as myocardin-related transcription factor-A (MRTF-A), and mediates gene transcription elicited by diverse signaling pathways. SRF/MRTF-A-dependent gene transcription is activated when nuclear MRTF-A levels increase, enabling the formation of transcriptionally active SRF/MRTF-A complexes. The level of nuclear MRTF-A is regulated by nuclear G-actin, which binds to MRTF-A and promotes its nuclear export. However, pathways that regulate nuclear actin levels are poorly understood. Here we show that MICAL-2, an atypical actin-regulatory protein, mediates SRF/MRTF-A-dependent gene transcription elicited by nerve growth factor and serum. MICAL-2 induces redox-dependent depolymerization of nuclear actin, which decreases nuclear G-actin and increases MRTF-A in the nucleus. Furthermore, we show that MICAL-2 is a target of CCG-1423, a small molecule inhibitor of SRF/MRTF-A-dependent transcription that exhibits efficacy in various preclinical disease models. These data identify redox modification of nuclear actin as a regulatory switch that mediates SRF/MRTF-A-dependent gene transcription.

Regulators of G protein signaling (RGS) proteins are key players in regulating signaling via G protein-coupled receptors. RGS proteins directly bind to the Gα-subunits of activated heterotrimeric G-proteins, and accelerate the rate of GTP hydrolysis, thereby rapidly deactivating G-proteins. Using atomistic simulations and NMR spectroscopy, we have studied in molecular detail the mechanism of action of CCG-50014, a potent small molecule inhibitor of RGS4 which covalently binds to cysteine residues on RGS4. We apply temperature-accelerated molecular dynamics (TAMD) to carry out enhanced conformational sampling of apo RGS4 structures, and consistently find that the α5-α6 helix pair of RGS4 can spontaneously span open-like conformations, allowing binding of CCG-50014 to the buried side-chain of Cys95. Both NMR experiments and MD simulations reveal chemical shift perturbations in residues in the vicinity of inhibitor binding site as well as in the RGS4-Gα binding interface. Consistent with a loss of G-protein binding, GAP activity, and allosteric mechanism of action of CCG-50014, our simulations of the RGS4-Gα complex in the presence of inhibitor suggest a relatively unstable protein-protein interaction. These results have potential implications for understanding how the conformational dynamics among RGS proteins may play a key role in the sensitivity of inhibitors.

BackgroundThe eye lens presents a unique opportunity to explore roles for specific molecules in cell proliferation, differentiation and development because cells remain in place throughout life and, like red blood cells and keratinocytes, they go through the most extreme differentiation, including removal of nuclei and cessation of protein synthesis. Ubiquitination controls many critical cellular processes, most of which require specific lysines on ubiquitin (Ub). Of the 7 lysines (K) least is known about effects of modification of K6.Methodology and Principal FindingsWe replaced K6 with tryptophan (W) because K6 is the most readily modified K and W is the most structurally similar residue to biotin. The backbone of K6W-Ub is indistinguishable from that of Wt-Ub. K6W-Ub is effectively conjugated and deconjugated but the conjugates are not degraded via the ubiquitin proteasome pathways (UPP). Expression of K6W-ubiquitin in the lens and lens cells results in accumulation of intracellular aggregates and also slows cell proliferation and the differentiation program, including expression of lens specific proteins, differentiation of epithelial cells into fibers, achieving proper fiber cell morphology, and removal of nuclei. The latter is critical for transparency, but the mechanism by which cell nuclei are removed has remained an age old enigma. This was also solved by expressing K6W-Ub. p27kip, a UPP substrate accumulates in lenses which express K6W-Ub. This precludes phosphorylation of nuclear lamin by the mitotic kinase, a prerequisite for disassembly of the nuclear membrane. Thus the nucleus remains intact and DNAseIIβ neither gains entry to the nucleus nor degrades the DNA. These results could not be obtained using chemical proteasome inhibitors that cannot be directed to specific tissues.Conclusions and SignificanceK6W-Ub provides a novel, genetic means to study functions of the UPP because it can be targeted to specific cells and tissues. A fully functional UPP is required to execute most stages of lens differentiation, specifically removal of cell nuclei. In the absence of a functional UPP, small aggregate prone, cataractous lenses are formed.