CX3CL1 (fractalkine) and CXCL16 are unique members of the chemokine family because they occur not only as soluble, but also as membrane-bound molecules. Expressed as type I transmembrane proteins, ...the ectodomain of both chemokines can be proteolytically cleaved from the cell surface, a process known as shedding. Our previous studies showed that the disintegrin and metalloproteinase 10 (ADAM10) mediates the largest proportion of constitutive CX3CL1 and CXCL16 shedding, but is not involved in the phorbolester-induced release of the soluble chemokines (inducible shedding). In this study, we introduce the calcium-ionophore ionomycin as a novel, very rapid, and efficient inducer of CX3CL1 and CXCL16 shedding. By transfection in COS-7 cells and ADAM10-deficient murine embryonic fibroblasts combined with the use of selective metalloproteinase inhibitors, we demonstrate that the inducible generation of soluble forms of these chemokines is dependent on ADAM10 activity. Analysis of the C-terminal cleavage fragments remaining in the cell membrane reveals multiple cleavage sites used by ADAM10, one of which is preferentially used upon stimulation with ionomycin. In adhesion studies with CX3CL1-expressing ECV-304 cells and cytokine-stimulated endothelial cells, we demonstrate that induced CX3CL1 shedding leads to the release of bound monocytic cell lines and PBMC from their cellular substrate. These data provide evidence for an inducible release mechanism via ADAM10 potentially important for leukocyte diapedesis.
Cajal bodies are nuclear structures involved in snRNP and snoRNP biogenesis, telomere maintenance and histone mRNA processing. Recently, the SUMO isopeptidase USPL1 was identified as a Cajal body ...component essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its relation to Cajal bodies. We demonstrate that the levels of RNAPII-transcribed snRNAs are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalizes with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus our data indicate that USPL1 plays a key role in the process of RNAPII-mediated snRNA transcription.
Mild sonication of eukaryotic cells produces native plasma membrane sheets that retain their docked organelles, cytoskeleton structures and cytoplasmic complexes. While the delicate organization of ...membranous protein complexes remains undisturbed, their inner plasmalemmel leaflet can be rapidly exposed to bathing solutions, enabling specific biochemical manipulations. Here, we apply this system to track membrane-biochemistry kinetics. We monitor soluble NSF-attachment protein receptor (SNARE) complex assembly and disassembly on the plasma membrane at high time resolution. The results suggest two-phase kinetics for the assembly process and dependence of the disassembly kinetics on both N-ethyl maleimide-sensitive factor (NSF) and soluble NSF-attachment protein (α-SNAP) concentrations.
The ATPase NSF (N-ethylmaleimide-sensitive factor) and its SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) cofactor constitute the ubiquitous enzymatic machinery responsible for ...recycling of the SNARE (SNAP receptor) membrane fusion machinery. The enzyme uses the energy of ATP hydrolysis to dissociate the constituents of the SNARE complex, which is formed during the fusion of a transport vesicle with the acceptor membrane. However, it is still unclear how NSF and the SNAP adaptor work together to take the tight SNARE bundle apart. SNAPs have been reported to attach to membranes independently from SNARE complex binding. We have investigated how efficient the disassembly of soluble and membrane-bound substrates are, comparing the two. We found that SNAPs support disassembly of membrane-bound SNARE complexes much more efficiently. Moreover, we identified a putative, conserved membrane attachment site in an extended loop within the N-terminal domain of α-SNAP. Mutation of two highly conserved, exposed phenylalanine residues on the extended loop prevent SNAPs from facilitating disassembly of membrane-bound SNARE complexes. This implies that the disassembly machinery is adapted to attack membrane-bound SNARE complexes, probably in their relaxed cis-configuration.
Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with ...the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.
This study characterizes USPL1—previously believed to be an ubiquitin protease—as a new type of SUMO isopeptidase and identifies the residues important for SUMO binding and cleavage. USPL1—but not its catalytic activity—is essential for cell proliferation.
The clinical relevance of comprehensive molecular analysis in rare cancers is not established. We analyzed the molecular profiles and clinical outcomes of 1,310 patients (rare cancers, 75.5%) ...enrolled in a prospective observational study by the German Cancer Consortium that applies whole-genome/exome and RNA sequencing to inform the care of adults with incurable cancers. On the basis of 472 single and six composite biomarkers, a cross-institutional molecular tumor board provided evidence-based management recommendations, including diagnostic reevaluation, genetic counseling, and experimental treatment, in 88% of cases. Recommended therapies were administered in 362 of 1,138 patients (31.8%) and resulted in significantly improved overall response and disease control rates (23.9% and 55.3%) compared with previous therapies, translating into a progression-free survival ratio >1.3 in 35.7% of patients. These data demonstrate the benefit of molecular stratification in rare cancers and represent a resource that may promote clinical trial access and drug approvals in this underserved patient population. SIGNIFICANCE: Rare cancers are difficult to treat; in particular, molecular pathogenesis-oriented medical therapies are often lacking. This study shows that whole-genome/exome and RNA sequencing enables molecularly informed treatments that lead to clinical benefit in a substantial proportion of patients with advanced rare cancers and paves the way for future clinical trials.
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Retrograde traffic between the Golgi apparatus and the endoplasmic reticulum (ER) is largely mediated by COPI-coated transport vesicles. In mammalian cells, retrograde traffic can pass through an ...intermediate compartment. Here, we report that the mammalian soluble
N-ethylmaleimide-sensitive factor (NSF) attachment receptor (SNARE) proteins mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 form a quaternary SNARE complex. Fluorescence resonance energy transfer (FRET) experiments prove that these interactions occur in the ER of living cells. In addition, mUse1/D12 and mSec20/BNIP1 form homo-oligomers in vivo. Furthermore, we show that mSec22b, mUse1/D12, mSec20/BNIP1, and syntaxin 18 are recruited into COPI-coated vesicles formed in vitro. Immunogold electron microscopy confirmed that these SNARE proteins colocalize with the KDEL receptor ERD2 in COPI-coated vesicles. Moreover, both FRET and immunoprecipitation experiments reveal interactions of these SNAREs with both ERD2 and COPI subunits. We conclude that the SNAREs described here are sorted via interaction with components of the COPI-dependent budding complex into Golgi-to-ER retrograde COPI vesicles and function in retrograde transport from the Golgi to the ER Golgi intermediate compartment (ERGIC) or the ER.
Cajal bodies are nuclear structures that are involved in biogenesis of snRNPs and snoRNPs, maintenance of telomeres and processing of histone mRNA. Recently, the SUMO isopeptidase USPL1 was ...identified as a component of Cajal bodies that is essential for cellular growth and Cajal body integrity. However, a cellular function for USPL1 is so far unknown. Here, we use RNAi-mediated knockdown in human cells in combination with biochemical and fluorescence microscopy approaches to investigate the function of USPL1 and its link to Cajal bodies. We demonstrate that levels of snRNAs transcribed by RNA polymerase (RNAP) II are reduced upon knockdown of USPL1 and that downstream processes such as snRNP assembly and pre-mRNA splicing are compromised. Importantly, we find that USPL1 associates directly with U snRNA loci and that it interacts and colocalises with components of the Little Elongation Complex, which is involved in RNAPII-mediated snRNA transcription. Thus, our data indicate that USPL1 plays a key role in RNAPII-mediated snRNA transcription.
Abstract
The benefit of molecularly-informed therapies in cancer of unknown primary (CUP) is unclear. Here, we use comprehensive molecular characterization by whole genome/exome, transcriptome and ...methylome analysis in 70 CUP patients to reveal substantial mutational heterogeneity with
TP53
,
MUC16
,
KRAS
,
LRP1B
and
CSMD3
being the most frequently mutated known cancer-related genes. The most common fusion partner is
FGFR2
, the most common focal homozygous deletion affects
CDKN2A
. 56/70 (80%) patients receive genomics-based treatment recommendations which are applied in 20/56 (36%) cases. Transcriptome and methylome data provide evidence for the underlying entity in 62/70 (89%) cases. Germline analysis reveals five (likely) pathogenic mutations in five patients. Recommended off-label therapies translate into a mean PFS ratio of 3.6 with a median PFS1 of 2.9 months (17 patients) and a median PFS2 of 7.8 months (20 patients). Our data emphasize the clinical value of molecular analysis and underline the need for innovative, mechanism-based clinical trials.
The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical ...efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug.
We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers.
Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio HR = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p < 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib.
A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic.
•Reliable predictors of pazopanib efficacy in soft-tissue sarcoma are lacking.•A gene expression-based score stratifies patients with sarcoma regarding pazopanib benefit.•The score is not predictive in patients with sarcoma who received other therapies.•The pazopanib efficacy predictor warrants prospective clinical investigation.
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