Identification of PLP2 and RAB5C as novel TPD52 binding partners through yeast two-hybrid screening

Tumor protein D52 (TPD52) is overexpressed in different cancers, but its molecular functions are poorly defined. A large, low-stringency yeast two-hybrid screen using full-length TPD52 bait identified known partners (TPD52, TPD52L1, TPD52L2, MAL2) and four other preys that reproducibly bound TPD52 and TPD52L1 baits (PLP2, RAB5C, GOLGA5, YIF1A). PLP2 and RAB5 interactions with TPD52 were confirmed in pull down assays, with interaction domain mapping experiments indicating that both proteins interact with a novel binding region of TPD52. This study provides insights into TPD52 functions, and ways to maximise the efficiency of low-stringency yeast two-hybrid screens.8 page(s

Correlation between pre-existing MEK1(P124) mutations and clinical and in vitro response to BRAF inhibitors in metastatic melanoma

Background: MEK1 mutations can confer resistance to BRAF inhibitors although pre-existing MEK1P¹²⁴ mutations do not preclude clinical responses to BRAF inhibitor therapy. We sought to determine if pre-existing MEK1P¹²⁴ mutations affected clinical outcome in BRAF inhibitor treated melanoma. Methods: Data from three published data sets, and from patients treated at our institutions, were analyzed to determine if pre-existing MEKP¹²⁴ mutations affect radiological response or progression-free survival (PFS) in BRAFV600 mutant metastatic melanoma patients treated with vemurafenib or dabrafenib. The effects of MEK1P¹²⁴ mutations on MAPK pathway activity and response to dabrafenib were also investigated in a series of cell models. Results: 123 patients with pre-treatment tumors tested for MEK1 mutations were included. Those with a pretreatment MEKP¹²⁴ mutation (n=12) had a poorer RECIST response (33% vs 71% CR or PR in MEK1P¹²⁴ vs MEK1 wild-type, p=0.008), this was associated with a shorter median PFS in those with a MEK1P¹²⁴ mutation (Median 3.1 vs 4.8 months, p=0.004). Introduction of MEK1 P124Q or P124S variants into BRAF-mutant SKMel28 melanoma cells resulted in diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival compared to cells ectopically expressing wild-type MEK1. The impact of MEK1P¹²⁴-variants was significantly less than the effect of MEK1K⁵⁷E, a known mechanism of acquired BRAF inhibitor resistance. Consistent with these data, two BRAF mutant cell lines with endogenous MEK1P¹²⁴ mutations, including a short term culture generated pre-treatment from a patient who responded poorly to combined dabrafenib and trametinib, showed weak sensitivity to dabrafenib (IC50s 21 and 26nM) compared to a panel of MEK1 wild type/BRAF mutant cell lines (median IC50 7nM; range 4-14nM). In contrast, melanoma cell lines showed equivalent sensitivity to ERK inhibition, irrespective of the MEK1 genotype. Conclusions: Pre-existing MEKP¹²⁴ mutations are associated with a reduced response to BRAF inhibitor therapy but are unlikely to affect response to ERK inhibitors.1 page(s

Regulation of GSK-3β and β-Catenin by Gαq in HEK293T cells

Recent studies have shown that heterotrimeric G proteins are involved in the regulation of the canonical Wnt/β-Catenin pathway. However, the mechanism(s) behind this involvement is (are) poorly understood. Our previous results have shown that activation of Gαq in Xenopus oocytes leads to inhibition of GSK-3β and stabilization of the β-Catenin protein, suggesting that Gαq might stabilize β-Catenin via inhibition of GSK-3β. In this study, we have observed similar results in HEK293T cells. In these cells optimal activation of endogenous Gαq by expressing M3-muscarinic acetylcholine receptor (with or without carbachol treatment), or exposing the cells to thrombin led to an increase of 2 to 3-fold in endogenous cytoplasmic β-Catenin protein levels. In addition, expression of the activated mutant of Gαq (GαqQL) dramatically enhanced accumulation of exogenous β-Catenin with no effect on β-catenin (CTNNB1) gene transcription. The Gαq-mediated cellular accumulation of β-Catenin was blocked by expression of a minigene encoding a Gαq specific inhibitory peptide but not by a minigene encoding a Gαs blocking peptide. Also, expression of GαqQL led to a significant reduction in GSK-3β kinase activity, supporting the idea that the positive role of Gαq signaling in inducing cellular accumulation of β-Catenin is mediated through inhibition of GSK-3β.6 page(s

Protein quality control and the amyotrophic lateral sclerosis/frontotemporal dementia continuum

Protein homeostasis, or proteostasis, has an important regulatory role in cellular function. Protein quality control mechanisms, including protein folding and protein degradation processes, have a crucial function in post-mitotic neurons. Cellular protein quality control relies on multiple strategies, including molecular chaperones, autophagy, the ubiquitin proteasome system, endoplasmic reticulum (ER)-associated degradation (ERAD) and the formation of stress granules (SGs), to regulate proteostasis. Neurodegenerative diseases are characterized by the presence of misfolded protein aggregates, implying that protein quality control mechanisms are dysfunctional in these conditions. Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are neurodegenerative diseases that are now recognized to overlap clinically and pathologically, forming a continuous disease spectrum. In this review article, we detail the evidence for dysregulation of protein quality control mechanisms across the whole ALS-FTD continuum, by discussing the major proteins implicated in ALS and/or FTD. We also discuss possible ways in which protein quality mechanisms could be targeted therapeutically in these disorders and highlight promising protein quality control-based therapeutics for clinical trials

Pathogenic mutation in the ALS/FTD gene, CCNF, causes elevated Lys48‑linked ubiquitylation and defective autophagy

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin-protein ligase (SCFcyclin F) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin-proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin FS621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin FWT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin FS621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin FS621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal-lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin FS621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis

Preexisting MEK1(P124) mutations diminish response to BRAF inhibitors in metastatic melanoma patients

Background: MEK1 mutations in melanoma can confer resistance to BRAF inhibitors, although preexisting MEK1P124 mutations do not preclude clinical responses. We sought to determine whether recurrent, preexisting MEK1P124 mutations affected clinical outcome in BRAF inhibitor–treated patients with melanoma. Methods: Data from four published datasets were analyzed to determine whether preexisting MEK1P124 mutations affect radiologic response or progression-free survival (PFS) in patients with BRAFV600-mutant metastatic melanoma treated with vemurafenib or dabrafenib. The effects of MEK1P124 mutations on MAPK pathway activity and response to BRAF inhibition were also investigated in a series of cell models. Results: In a pooled analysis of 123 patients, the presence of a pretreatment MEK1P124 mutation (N = 12, 10%) was associated with a poorer RECIST response (33% vs. 72% in MEK1P124Q/S vs. MEK1P124 wild-type, P = 0.018), and a shorter PFS (median 3.1 vs. 4.8 months, P = 0.004). Furthermore, MEK1P124Q/S mutations were shown to have independent kinase activity and introduction of these mutations into a BRAF-mutant melanoma cell line diminished inhibition of ERK phosphorylation by dabrafenib and enhanced clonogenic survival in the presence of dabrafenib compared with cells ectopically expressing wild-type MEK1. Consistent with these data, two BRAF-mutant cell lines with endogenous MEK1P124 mutations showed intermediate sensitivity to dabrafenib, but were highly sensitive to downstream inhibition of MEK or ERK. Conclusion: Taken together, our data indicate that preexisting MEK1P124 mutations are associated with a reduced response to BRAF inhibitor therapy and identify a subset of patients with BRAF-mutant melanoma likely to benefit from combination therapies involving MEK or ERK inhibitors.8 page(s

The Formin INF2 regulates basolateral-to-apical transcytosis and lumen formation in association with Cdc42 and MAL2

Transcytosis is a widespread pathway for apical targeting in epithelial cells. MAL2, an essential protein of the machinery for apical transcytosis, functions by shuttling in vesicular carriers between the apical zone and the cell periphery. We have identified INF2, an atypical formin with actin polymerization and depolymerization activities, which is a binding partner of MAL2. MAL2-positive vesicular carriers associate with short actin filaments during transcytosis in a process requiring INF2. INF2 binds Cdc42 in a GTP-loaded-dependent manner. Cdc42 and INF2 regulate MAL2 dynamics and are necessary for apical transcytosis and the formation of lateral lumens in hepatoma HepG2 cells. INF2 and MAL2 are also essential for the formation of the central lumen in organotypic cultures of epithelial MDCK cells. Our results reveal a functional mechanism whereby Cdc42, INF2, and MAL2 are sequentially ordered in a pathway dedicated to the regulation of transcytosis and lumen formation.14 page(s

Increased MAPK reactivation in early resistance to dabrafenib/trametinib combination therapy of BRAF-mutant metastatic melanoma

One-third of BRAF-mutant metastatic melanoma patients treated with combined BRAF and MEK inhibition progress within 6 months. Treatment options for these patients remain limited. Here we analyse 20 BRAF V600-mutant melanoma metastases derived from 10 patients treated with the combination of dabrafenib and trametinib for resistance mechanisms and genetic correlates of response. Resistance mechanisms are identified in 9/11 progressing tumours and MAPK reactivation occurred in 9/10 tumours, commonly via BRAF amplification and mutations activating NRAS and MEK2. Our data confirming that MEK2 C125S, but not the synonymous MEK1 C121S protein, confers resistance to combination therapy highlight the functional differences between these kinases and the preponderance of MEK2 mutations in combination therapy-resistant melanomas. Exome sequencing did not identify additional progression-specific resistance candidates. Nevertheless, most melanomas carried additional oncogenic mutations at baseline (for example, RAC1 and AKT3) that activate the MAPK and PI3K pathways and are thus predicted to diminish response to MAPK inhibitors.9 page(s