Reduced Protein Stability of 11 Pathogenic Missense STXBP1/MUNC18-1 Variants and Improved Disease Prediction.

BACKGROUND: Pathogenic variants in STXBP1/MUNC18-1 cause severe encephalopathies that are among the most common in genetic neurodevelopmental disorders. Different molecular disease mechanisms have been proposed, and pathogenicity prediction is limited. In this study, we aimed to define a generalized disease concept for STXBP1-related disorders and improve prediction. METHODS: A cohort of 11 disease-associated and 5 neutral variants (detected in healthy individuals) were tested in 3 cell-free assays and in heterologous cells and primary neurons. Protein aggregation was tested using gel filtration and Triton X-100 insolubility. PRESR (predicting STXBP1-related disorder), a machine learning algorithm that uses both sequence- and 3-dimensional structure-based features, was developed to improve pathogenicity prediction using 231 known disease-associated variants and comparison to our experimental data. RESULTS: Disease-associated variants, but none of the neutral variants, produced reduced protein levels. Cell-free assays demonstrated directly that disease-associated variants have reduced thermostability, with most variants denaturing around body temperature. In addition, most disease-associated variants impaired SNARE-mediated membrane fusion in a reconstituted assay. Aggregation/insolubility was observed for none of the variants in vitro or in neurons. PRESR outperformed existing tools substantially: Matthews correlation coefficient = 0.71 versus <0.55. CONCLUSIONS: These data establish intrinsic protein instability as the generalizable, primary cause for STXBP1-related disorders and show that protein-specific ortholog and 3-dimensional information improve disease prediction. PRESR is a publicly available diagnostic tool.

Never-homozygous genetic variants in healthy populations are potential recessive disease candidates.

The rapid pace with which genetic variants are now being determined means there is a pressing need to understand how they affect biological systems. Variants from healthy individuals have previously been used to study blood groups or HLA diversity and to identify genes that can apparently be nonfunctional in healthy people. These studies and others have observed a lower than expected frequency of homozygous individuals for potentially deleterious alleles, which would suggest that several of these alleles can lead to recessive disorders. Here we exploited this principle to hunt for potential disease variants in genomes from healthy people. We identified at least 108 exclusively heterozygous variants with evidence for an impact on biological function. We discuss several examples of candidate variants/genes including CCDC8, PANK3, RHD and NLRP12. Overall, the results suggest there are many, comparatively frequent, potentially lethal or disease-causing variants lurking in healthy human populations.

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes.

Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37 °C to 39 °C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.

Inhibition of CD95/CD95L (FAS/FASLG) Signaling with APG101 Prevents Invasion and Enhances Radiation Therapy for Glioblastoma.

CD95 (Fas/APO-1), a death receptor family member, activity has been linked to tumorigenicity in multiple cancers, including glioblastoma multiforme (GBM). A phase II clinical trial on relapsed glioblastoma patients demonstrated that targeted inhibition of CD95 signaling via the CD95 ligand (CD95L) binding and neutralizing Fc-fusion protein APG101 (asunercept) prolonged patient survival. Although CD95 signaling may be relevant for multiple aspects of tumor growth, the mechanism of action of APG101 in glioblastoma is not clear. APG101 action was examined by in vitro proliferation, apoptosis, and invasion assays with human and murine glioma and human microglial cells, as well as in vivo therapy studies with orthotopic gliomas and clinical data. APG101 inhibits CD95L-mediated invasion of glioma cells. APG101 treatment was effective in glioma-bearing mice, independently of the presence or absence of CD4 and CD8 T lymphocytes, which should be sensitive to CD95L. Combined with radiotherapy, APG101 demonstrated a reduction of tumor growth, fewer tumor satellites, reduced activity of matrix metalloproteinases (MMP) as well as prolonged survival of tumor-bearing mice compared with radiotherapy alone. Inhibiting rather than inducing CD95 activity is a break-of-paradigm therapeutic approach for malignant gliomas. Evidence, both in vitro and in vivo, is provided that CD95L-binding fusion protein treatment enhanced the efficacy of radiotherapy and reduced unwanted proinfiltrative effects by reducing metalloproteinase activity by directly affecting the tumor cells.Implications: APG101 (asunercept) successfully used in a controlled phase II glioblastoma trial (NCT01071837) acts anti-invasively by inhibiting matrix metalloproteinase signaling, resulting in additive effects together with radiotherapy and helping to further develop a treatment for this devastating disease. Mol Cancer Res; 16(5); 767-76. ©2018 AACR.

Tweety-Homolog 1 Drives Brain Colonization of Gliomas.

Early and progressive colonization of the healthy brain is one hallmark of diffuse gliomas, including glioblastomas. We recently discovered ultralong (>10 to hundreds of microns) membrane protrusions [tumor microtubes (TMs)] extended by glioma cells. TMs have been associated with the capacity of glioma cells to effectively invade the brain and proliferate. Moreover, TMs are also used by some tumor cells to interconnect to one large, resistant multicellular network. Here, we performed a correlative gene-expression microarray and in vivo imaging analysis, and identified novel molecular candidates for TM formation and function. Interestingly, these genes were previously linked to normal CNS development. One of the genes scoring highest in tests related to the outgrowth of TMs was tweety-homolog 1 (TTYH1), which was highly expressed in a fraction of TMs in mice and patients. Ttyh1 was confirmed to be a potent regulator of normal TM morphology and of TM-mediated tumor-cell invasion and proliferation. Glioma cells with one or two TMs were mainly responsible for effective brain colonization, and Ttyh1 downregulation particularly affected this cellular subtype, resulting in reduced tumor progression and prolonged survival of mice. The remaining Ttyh1-deficient tumor cells, however, had more interconnecting TMs, which were associated with increased radioresistance in those small tumors. These findings imply a cellular and molecular heterogeneity in gliomas regarding formation and function of distinct TM subtypes, with multiple parallels to neuronal development, and suggest that Ttyh1 might be a promising target to specifically reduce TM-associated brain colonization by glioma cells in patients.SIGNIFICANCE STATEMENT In this report, we identify tweety-homolog 1 (Ttyh1), a membrane protein linked to neuronal development, as a potent driver of tumor microtube (TM)-mediated brain colonization by glioma cells. Targeting of Ttyh1 effectively inhibited the formation of invasive TMs and glioma growth, but increased network formation by intercellular TMs, suggesting a functional and molecular heterogeneity of the recently discovered TMs with potential implications for future TM-targeting strategies.

Evaluation of dipeptide nitriles as inhibitors of rhodesain, a major cysteine protease of Trypanosoma brucei.

A series of dipeptide nitriles known as inhibitors of mammalian cathepsins were evaluated for inhibition of rhodesain, the cathepsin L-like protease of Trypanosoma brucei. Compound 35 consisting of a Leu residue fitting into the S2 pocket and a triarylic moiety consisting of thiophene, a 1,2,4-oxadiazole and a phenyl ring fitting into the S3 pocket, and compound 33 with a 3-bromo-Phe residue (S2) and a biphenyl fragment (S3) were found to inhibit rhodesain in the single-digit nanomolar range. The observed steep structure-activity relationship could be explained by covalent docking simulations. With their high selectivity indices (ca. 200) and the good antitrypanosomal activity (8µM) the compounds represent promising starting points for new rhodesain inhibitors.

Brain tumour cells interconnect to a functional and resistant network.

Astrocytic brain tumours, including glioblastomas, are incurable neoplasms characterized by diffusely infiltrative growth. Here we show that many tumour cells in astrocytomas extend ultra-long membrane protrusions, and use these distinct tumour microtubes as routes for brain invasion, proliferation, and to interconnect over long distances. The resulting network allows multicellular communication through microtube-associated gap junctions. When damage to the network occurred, tumour microtubes were used for repair. Moreover, the microtube-connected astrocytoma cells, but not those remaining unconnected throughout tumour progression, were protected from cell death inflicted by radiotherapy. The neuronal growth-associated protein 43 was important for microtube formation and function, and drove microtube-dependent tumour cell invasion, proliferation, interconnection, and radioresistance. Oligodendroglial brain tumours were deficient in this mechanism. In summary, astrocytomas can develop functional multicellular network structures. Disconnection of astrocytoma cells by targeting their tumour microtubes emerges as a new principle to reduce the treatment resistance of this disease.