Breast tumours are embedded in a collagen I-rich extracellular matrix (ECM) network, where nutrients are scarce due to limited blood flow and elevated tumour growth. Metabolic adaptation is required ...for cancer cells to endure these conditions. Here, we demonstrated that the presence of ECM supported the growth of invasive breast cancer cells, but not non-transformed mammary epithelial cells, under amino acid starvation, through a mechanism that required macropinocytosis-dependent ECM uptake. Importantly, we showed that this behaviour was acquired during carcinoma progression. ECM internalisation, followed by lysosomal degradation, contributed to the up-regulation of the intracellular levels of several amino acids, most notably tyrosine and phenylalanine. This resulted in elevated tyrosine catabolism on ECM under starvation, leading to increased fumarate levels, potentially feeding into the tricarboxylic acid (TCA) cycle. Interestingly, this pathway was required for ECM-dependent cell growth and invasive cell migration under amino acid starvation, as the knockdown of p-hydroxyphenylpyruvate hydroxylase-like protein (HPDL), the third enzyme of the pathway, opposed cell growth and motility on ECM in both 2D and 3D systems, without affecting cell proliferation on plastic. Finally, high HPDL expression correlated with poor prognosis in breast cancer patients. Collectively, our results highlight that the ECM in the tumour microenvironment (TME) represents an alternative source of nutrients to support cancer cell growth by regulating phenylalanine and tyrosine metabolism.
Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic ...checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain 'locked' onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.
Calcium-dependent protein kinases (CDPKs) play key regulatory roles in the life cycle of the malaria parasite, but in many cases their precise molecular functions are unknown. Using the rodent ...malaria parasite Plasmodium berghei, we show that CDPK1, which is known to be essential in the asexual blood stage of the parasite, is expressed in all life stages and is indispensable during the sexual mosquito life-cycle stages. Knockdown of CDPK1 in sexual stages resulted in developmentally arrested parasites and prevented mosquito transmission, and these effects were independent of the previously proposed function for CDPK1 in regulating parasite motility. In-depth translational and transcriptional profiling of arrested parasites revealed that CDPK1 translationally activates mRNA species in the developing zygote that in macrogametes remain repressed via their 3′ and 5′UTRs. These findings indicate that CDPK1 is a multifunctional protein that translationally regulates mRNAs to ensure timely and stage-specific protein expression.
► CDPK1 is an abundant kinase of invasive and noninvasive life-cycle stages ► It functions in gametocyte emergence and zygote-to-ookinete transformation ► CDPK1 mediates transcriptional activation of some silenced mRNAs in the zygote ► CDPK1 is crucial for the mosquito transmission of P. berghei
Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While ...proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid-liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.
Using a comprehensive proteomic approach, Schwenk et al. (2014), in this issue of Neuron, resolve the differential composition of AMPA receptor complexes in brain regions and through development. ...This work reveals a specificity in AMPA receptor complex assembly that is dynamic in both space and time.
Fas plays a major role in regulating ligand-induced apoptosis in many cell types. It is well known that several cancers demonstrate reduced cell surface levels of Fas and thus escape a potential ...control system via ligand-induced apoptosis, although underlying mechanisms are unclear. Here we report that the endosome associated trafficking regulator 1 (ENTR1), controls cell surface levels of Fas and Fas-mediated apoptotic signalling. ENTR1 regulates, via binding to the coiled coil domain protein Dysbindin, the delivery of Fas from endosomes to lysosomes thereby controlling termination of Fas signal transduction. We demonstrate that ENTR1 is cleaved during Fas-induced apoptosis in a caspase-dependent manner revealing an unexpected interplay of apoptotic signalling and regulation of endolysosomal trafficking resulting in a positive feedback signalling-loop. Our data provide insights into the molecular mechanism of Fas post-endocytic trafficking and signalling, opening possible explanations on how cancer cells regulate cell surface levels of death receptors.
Characterization of the composition of the postsynaptic proteome (PSP) provides a framework for understanding the overall organization and function of the synapse in normal and pathological ...conditions. We have identified 698 proteins from the postsynaptic terminal of mouse CNS synapses using a series of purification strategies and analysis by liquid chromatography tandem mass spectrometry and large‐scale immunoblotting. Some 620 proteins were found in purified postsynaptic densities (PSDs), nine in AMPA‐receptor immuno‐purifications, 100 in isolates using an antibody against the NMDA receptor subunit NR1, and 170 by peptide‐affinity purification of complexes with the C‐terminus of NR2B. Together, the NR1 and NR2B complexes contain 186 proteins, collectively referred to as membrane‐associated guanylate kinase‐associated signalling complexes. We extracted data from six other synapse proteome experiments and combined these with our data to provide a consensus on the composition of the PSP. In total, 1124 proteins are present in the PSP, of which 466 were validated by their detection in two or more studies, forming what we have designated the Consensus PSD. These synapse proteome data sets offer a basis for future research in synaptic biology and will provide useful information in brain disease and mental disorder studies.
Dysregulated neutrophilic inflammation can be highly destructive in chronic inflammatory diseases due to prolonged neutrophil lifespan and continual release of histotoxic mediators in inflamed ...tissues. Therapeutic induction of neutrophil apoptosis, an immunologically silent form of cell death, may be beneficial in these diseases, provided that the apoptotic neutrophils are efficiently cleared from the tissue. Previous research in our group identified ErbB inhibitors as able to induce neutrophil apoptosis and reduce neutrophilic inflammation both
and
. Here, we extend that work using a clinical ErbB inhibitor, neratinib, which has the potential to be repurposed in inflammatory diseases. We show that neratinib reduces neutrophilic migration o an inflammatory site in zebrafish larvae. Neratinib upregulates efferocytosis and reduces the number of persisting neutrophil corpses in mouse models of acute, but not chronic, lung injury, suggesting that the drug may have therapeutic benefits in acute inflammatory settings. Phosphoproteomic analysis of human neutrophils shows that neratinib modifies the phosphorylation of proteins regulating apoptosis, migration, and efferocytosis. This work identifies a potential mechanism for neratinib in treating acute lung inflammation by upregulating the clearance of dead neutrophils and, through examination of the neutrophil phosphoproteome, provides important insights into the mechanisms by which this may be occurring.
Phosphorylation, the most intensively studied and common PTM on proteins, is a complex biological phenomenon. Its complexity manifests itself in the large numbers of proteins that attach it, remove ...it and recognise it as a protein code. Since the first report of protein phosphorylation on vitellin 100 years ago, a wide variety of biochemical and analytical chemical approaches have been developed to enrich and detect protein phosphorylation. The last 5 years have witnessed a renaissance in methodologies capable of characterising protein phosphorylation on a proteome-scale. These technological advances have allowed identification of hundreds to thousands of phosphorylation sites in a proteome and have resulted in a profound paradigm shift. For the first time, using quantitative MS, the topology and significance of global phosphorylation networks may be investigated, marking a new era of cell signalling research. This review addresses recent technological advances in the purification of phosphorylated proteins and peptides and current MS-based strategies used to qualitatively and quantitatively probe these enriched phosphoproteomes. In addition, we review the application of complementary array-based technologies to derive signalling networks from kinase-substrate interactions and discuss future challenges in the field.
Arc is an activity-regulated neuronal protein, but little is known about its interactions, assembly into multiprotein complexes, and role in human disease and cognition. We applied an integrated ...proteomic and genetic strategy by targeting a tandem affinity purification (TAP) tag and Venus fluorescent protein into the endogenous Arc gene in mice. This allowed biochemical and proteomic characterization of native complexes in wild-type and knockout mice. We identified many Arc-interacting proteins, of which PSD95 was the most abundant. PSD95 was essential for Arc assembly into 1.5-MDa complexes and activity-dependent recruitment to excitatory synapses. Integrating human genetic data with proteomic data showed that Arc-PSD95 complexes are enriched in schizophrenia, intellectual disability, autism, and epilepsy mutations and normal variants in intelligence. We propose that Arc-PSD95 postsynaptic complexes potentially affect human cognitive function.
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•TAP tag and purification of endogenous Arc protein complexes from the mouse brain•PSD95 is the major Arc binding protein, and both assemble into 1.5-MDa supercomplexes•PSD95 is essential for recruitment of Arc to synapses•Mutations and genetic variants in Arc-PSD95 are linked to cognition
Fernández et al. use genetics and proteomics to study the Arc protein in the mouse brain. PSD95 recruits Arc to the synapse and assembles it into signaling complexes with neurotransmitter receptors and other proteins. Arc-PSD95 supercomplexes contain genetic variants previously linked to epilepsy, schizophrenia, intellectual disability, and IQ.
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