Colorectal cancer (CRC) remains a major cause of cancer related-death in developed countries. The mortality risk is correlated with the stage of CRC determined at the primary diagnosis and early ...diagnosis is associated with enhanced survival rate. Currently, only faecal occult blood tests are used to screen for CRC. Consequently, there is an incentive to identify specific markers of CRC. We used quantitative proteomic analysis of serum samples to characterize protein profiles in adenoma, CRC and healthy control samples. We identified 89 distinct proteins modulated between normal, colorectal adenoma and carcinoma patients. This list emphasizes proteins involved in enzyme regulator activities and in particular the serpin family. In serum samples, protein profiles of three members of the serpin family (SERPINA1, SERPINA3 and SERPINC1) were confirmed by ELISA assays. We obtained sensitivity/specificity values of 95%/95% for both SERPINA1 and SERPINC1, and 95%/55% for SERPINA3. This study supports the idea that serum proteins can discriminate adenoma and CRC patients from unaffected patients and reveals a panel of regulated proteins that might be useful for selecting patients for colonoscopy. By evaluating SERPINA1, SERPINA3 and SERPINC1, we highlight the potential role of the serpin family during the development and progression of CRC.
Colorectal cancer (CRC) remains a major cause of cancer mortality throughout the world. However, very few CRC biomarkers have satisfactory sensitivity and specificity in clinical practice. To the best of our knowledge our study is the first to profile sera proteomes between adenoma, CRC and healthy patients. We report a comprehensive list of proteins that may be used as early diagnostic biomarkers of CRC. It is noteworthy that 17% of these modulated proteins have been previously described as candidate biomarkers in CRC. Enzyme regulator activity was found to be the main molecular function among these proteins and, in particular, there was an enrichment of members of the serpin family. The subsequent verification on a new cohort by ELISA demonstrates that these serpins could be useful to discriminate healthy from colorectal carcinoma patients with a high sensitivity and specificity. The combination of these biomarkers should increase predictive powers of CRC diagnosis. The remaining candidates form a reserve for further evaluation of additional biomarkers for CRC diagnosis.
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•First study profiling proteome between adenoma, CRC and healthy patients•Quantitative proteomics revealed 89 distinct proteins modulated between groups•Several of these modulated proteins have been recently described as implicated in CRC•Enzyme regulator activity was found to be the main molecular function•One third of the modulated proteins are members of the serpin family•SERPINA1, A3 and C1 have been highlighted as candidate biomarkers for CRC diagnosis
Interactions between bacterial and fungal cells shape many polymicrobial communities. Bacteria elaborate diverse strategies to interact and compete with other organisms, including the deployment of ...protein secretion systems. The type VI secretion system (T6SS) delivers toxic effector proteins into host eukaryotic cells and competitor bacterial cells, but, surprisingly, T6SS-delivered effectors targeting fungal cells have not been reported. Here we show that the 'antibacterial' T6SS of Serratia marcescens can act against fungal cells, including pathogenic Candida species, and identify the previously undescribed effector proteins responsible. These antifungal effectors, Tfe1 and Tfe2, have distinct impacts on the target cell, but both can ultimately cause fungal cell death. 'In competition' proteomics analysis revealed that T6SS-mediated delivery of Tfe2 disrupts nutrient uptake and amino acid metabolism in fungal cells, and leads to the induction of autophagy. Intoxication by Tfe1, in contrast, causes a loss of plasma membrane potential. Our findings extend the repertoire of the T6SS and suggest that antifungal T6SSs represent widespread and important determinants of the outcome of bacterial-fungal interactions.
In this study, spatiotemporal changes of the phagosomal proteome in dendritic cells are investigated by label-free, quantitative mass spectrometry to characterize phagosomal maturation in resting and ...LPS-stimulated cells. Phagosomes of LPS-treated cells show a decreased recruitment of proteins involved in phagosome maturation but have an increased recruitment of proteins involved in antigen cross-presentation. Hierarchical clustering demonstrated that the acquisition of proteins to phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs.
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Highlights
•Characterization of the phagosomal proteome comparing resting and LPS-treated BMDCs.•Label-free quantification determined 2843 phagosomal proteins.•Reduced recruitment of hydrolases and V-ATPase to phagosomes of LPS-treated cells.•Increased recruitment of antigen cross-presentation molecules to these phagosomes.
Dendritic cells (DCs) are professional phagocytes that use innate sensing and phagocytosis to internalize and degrade self as well as foreign material, such as pathogenic bacteria, within phagosomes. These intracellular compartments are equipped to generate antigenic peptides that serve as source for antigen presentation to T cells initiating adaptive immune responses. The phagosomal proteome of DCs is only partially studied and is highly dynamic as it changes during phagosome maturation, when phagosomes sequentially interact with endosomes and lysosomes. In addition, the activation status of the phagocyte can modulate the phagosomal composition and is able to shape phagosomal functions. In this study, we determined spatiotemporal changes of the proteome of DC phagosomes during their maturation and compared resting and lipopolysaccharide (LPS)-stimulated bone marrow-derived DCs by label-free, quantitative mass spectrometry. Ovalbumin-coupled latex beads were used as phagocytosis model system and revealed that LPS-treated DCs show decreased recruitment of proteins involved in phagosome maturation, such as subunits of the vacuolar proton ATPase, cathepsin B, D, S, and RAB7. In contrast, those phagosomes were characterized by an increased recruitment of proteins involved in antigen cross-presentation, e.g. different subunits of MHC I molecules, the proteasome and tapasin, confirming the observed increase in cross-presentation efficacy in those cells. Further, several proteins were identified that were not previously associated with phagosomal functions. Hierarchical clustering of phagosomal proteins demonstrated that their acquisition to DC phagosomes is not only dependent on the duration of phagosome maturation but also on the activation state of DCs. Thus, our study provides a comprehensive overview of how DCs alter their phagosome composition in response to LPS, which has profound impact on the initiation of efficient immune responses.
Ubiquitin is post-translationally modified by phosphorylation at several sites, but the consequences of these modifications are largely unknown. Here, we synthesize multi-milligram quantities of ...ubiquitin phosphorylated at serine 20, serine 57, and serine 65 via genetic code expansion. We use these phosphoubiquitins for the enzymatic assembly of 20 isomeric phosphoubiquitin dimers, with different sites of isopeptide linkage and/or phosphorylation. We discover that phosphorylation of serine 20 on ubiquitin converts UBE3C from a dual-specificity E3 ligase into a ligase that primarily synthesizes K48 chains. We profile the activity of 31 deubiquitinases on the isomeric phosphoubiquitin dimers in 837 reactions, and we discover that phosphorylation at distinct sites in ubiquitin can activate or repress cleavage of a particular linkage by deubiquitinases and that phosphorylation at a single site in ubiquitin can control the specificity of deubiquitinases for distinct ubiquitin linkages.
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•Milligram quantities of ubiquitin phosphorylated at Ser 20, 57, or 65 are purified•Twenty isomeric phosphoubiquitin dimers are assembled and purified•UBE3C chain synthesis specificity is controlled by Ser 20 ubiquitin phosphorylation•Phosphorylation of ubiquitin controls deubiquitinase activity and linkage specificity
Huguenin-Dezot et al. combine genetic code expansion and enzymatic assembly to synthesize 20 isomeric phosphoubiquitin chains with distinct Ser phosphorylation and/or isopeptide linkage sites. They discover that ubiquitin phosphorylation can control E3 ligase specificity and deubiquitinase specificity.
The Type VI secretion system (T6SS) is widely used by bacterial pathogens as an effective weapon against bacterial competitors and is also deployed against host eukaryotic cells in some cases. It is ...a contractile nanomachine which delivers toxic effector proteins directly into target cells by dynamic cycles of assembly and firing. Bacterial cells adopt distinct post-translational regulatory strategies for deployment of the T6SS. 'Defensive' T6SSs assemble and fire in response to incoming attacks from aggressive neighbouring cells, and can utilise the Threonine Protein Phosphorylation (TPP) regulatory pathway to achieve this control. However, many T6SSs are 'offensive', firing at all-comers without the need for incoming attack or other cell contact-dependent signal. Post-translational control of the offensive mode has been less well defined but can utilise components of the same TPP pathway. Here, we used the anti-bacterial T6SS of Serratia marcescens to elucidate post-translational regulation of offensive T6SS deployment, using single-cell microscopy and genetic analyses. We show that the integration of the TPP pathway with the negative regulator TagF to control core T6SS machine assembly is conserved between offensive and defensive T6SSs. Signal-dependent PpkA-mediated phosphorylation of Fha is required to overcome inhibition of membrane complex assembly by TagF, whilst PppA-mediated dephosphorylation promotes spatial reorientation and efficient killing. In contrast, the upstream input of the TPP pathway defines regulatory strategy, with a new periplasmic regulator, RtkS, shown to interact with the PpkA kinase in S. marcescens. We propose a model whereby the opposing actions of the TPP pathway and TagF impose a delay on T6SS re-assembly after firing, providing an opportunity for spatial re-orientation of the T6SS in order to maximise the efficiency of competitor cell targeting. Our findings provide a better understanding of how bacterial cells deploy competitive weapons effectively, with implications for the structure and dynamics of varied polymicrobial communities.
Background Mutations in the X-linked gene cyclin-dependent kinase-like 5 (CDKL5) cause a severe neurological disorder characterised by early-onset epileptic seizures, autism and intellectual ...disability (ID). Impaired hippocampal function has been implicated in other models of monogenic forms of autism spectrum disorders and ID and is often linked to epilepsy and behavioural abnormalities. Many individuals with CDKL5 deficiency disorder (CDD) have null mutations and complete loss of CDKL5 protein, therefore in the current study we used a Cdkl5.sup.-/y rat model to elucidate the impact of CDKL5 loss on cellular excitability and synaptic function of CA1 pyramidal cells (PCs). We hypothesised abnormal pre and/or post synaptic function and plasticity would be observed in the hippocampus of Cdkl5.sup.-/y rats. Methods To allow cross-species comparisons of phenotypes associated with the loss of CDKL5, we generated a loss of function mutation in exon 8 of the rat Cdkl5 gene and assessed the impact of the loss of CDLK5 using a combination of extracellular and whole-cell electrophysiological recordings, biochemistry, and histology. Results Our results indicate that CA1 hippocampal long-term potentiation (LTP) is enhanced in slices prepared from juvenile, but not adult, Cdkl5.sup.-/y rats. Enhanced LTP does not result from changes in NMDA receptor function or subunit expression as these remain unaltered throughout development. Furthermore, Ca.sup.2+ permeable AMPA receptor mediated currents are unchanged in Cdkl5.sup.-/y rats. We observe reduced mEPSC frequency accompanied by increased spine density in basal dendrites of CA1 PCs, however we find no evidence supporting an increase in silent synapses when assessed using a minimal stimulation protocol in slices. Additionally, we found no change in paired-pulse ratio, consistent with normal release probability at Schaffer collateral to CA1 PC synapses. Conclusions Our data indicate a role for CDKL5 in hippocampal synaptic function and raise the possibility that altered intracellular signalling rather than synaptic deficits contribute to the altered plasticity. Limitations This study has focussed on the electrophysiological and anatomical properties of hippocampal CA1 PCs across early postnatal development. Studies involving other brain regions, older animals and behavioural phenotypes associated with the loss of CDKL5 are needed to understand the pathophysiology of CDD. Keywords: CDKL5, rat, hippocampus, synaptic plasticity, intrinsic properties, AMPA receptor, NMDA receptor
Colorectal cancer (CRC) remains a major cause of cancer fatalities in developed countries. The risk of death is correlated to the stage of CRC during the primary diagnosis. Early diagnosis is closely ...associated with enhanced survival rate. We therefore investigated the AP-F13A1 as a potential protein marker of CRC.
The protein expression of FXIII in 40 serum samples was evaluated by enzyme-linked immunosorbent assays. Additionally, targeted proteomic assays (LC-PRM) were used to evaluate the expression of the activation peptide of F13A1 (AP-F13A1) in a further 113 serum samples. Results were analyzed by the Wilcoxon test and receiver operating characteristic curves generated to assess statistical differences and diagnostic factors between CRC patients and controls.
AP-F13A1 was quantified in human serum samples using calibration curves with excellent linearity. AP-F13A1 was reduced in CRC patients using PRM assays from two distinct biobanks. The AUC for AP-F13A1 were 0.95 and 0.93. Sensitivity/specificity values for the two sets of patients were 75%/95% and 71%/95% respectively.
We have presented the proof of principle that in vivo release of AP-F13A1 can be measured by PRM-based strategies in CRC serum samples. AP-F13A1 may be an effective serological biomarker as part of a screening program of CRC detection.
Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype ...that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells.
Synopsis
The breast cancer drug palbociclib arrests cells in G1 phase by CDK4/6 inhibition, but also causes cellular senescence. Thermal proteome profiling shows that the latter is mediated by increased proteasome activation through reduced ECM29 binding.
Mass spectrometry‐based cellular thermal shift assay (MS‐CeTSA) analysis of CDK4/6 inhibitor palbociclib targets in MCF7 human breast cancer cells identifies protein complexes including the 20S proteasome.
Palbociclib treatment increases proteasome activity independently of the ubiquitin pathway.
Palbociclib activates the proteasome by disassociating the proteolysis‐inhibiting scaffold protein ECM29.
Proteasome activation is required for palbociclib‐induced cellular senescence, which is sensitive to the proteasome inhibitor bortezomib.
The proteasome is a novel downstream target for the anti‐cancer drug palbociclib inhibiting proliferation by reduced proteasomal association with its inhibitor EMC29.
Mutations in the leucine‐rich repeat kinase 2 (LRRK2) are associated with Parkinson's disease, chronic inflammation and mycobacterial infections. Although there is evidence supporting the idea that ...LRRK2 has an immune function, the cellular function of this kinase is still largely unknown. By using genetic, pharmacological and proteomics approaches, we show that LRRK2 kinase activity negatively regulates phagosome maturation via the recruitment of the Class III phosphatidylinositol‐3 kinase complex and Rubicon to the phagosome in macrophages. Moreover, inhibition of LRRK2 kinase activity in mouse and human macrophages enhanced Mycobacterium tuberculosis phagosome maturation and mycobacterial control independently of autophagy. In vivo, LRRK2 deficiency in mice resulted in a significant decrease in M. tuberculosis burdens early during the infection. Collectively, our findings provide a molecular mechanism explaining genetic evidence linking LRRK2 to mycobacterial diseases and establish an LRRK2‐dependent cellular pathway that controls M. tuberculosis replication by regulating phagosome maturation.
Synopsis
Possible immune functions of LRRK2, a kinase frequently mutated in Parkinson's disease, have remained ill‐defined. Genetic, pharmacological and proteomics approaches now reveal it as a negative regulator of phagosome maturation in macrophages, thereby affecting the control of Mycobacterium tuberculosis (Mtb) infection.
LRRK2 loss targets Mtb to phagolysosomes and limits Mtb replication.
LRRK2 inhibition enhances phagosome maturation in macrophages.
LRRK2 activity is required for the recruitment of class III PI3K/Rubicon into phagosomes.
Loss of LRRK2 enhances innate immunity to Mtb in mice.
LRRK2 KO alters inflammatory profiles after Mtb infection in vitro and in vivo.
The Parkinson's disease‐associated kinase LRRK2 affects innate immune control of mycobacterial infections in vitro and in vivo via phagosome recruitment of class III PI3K and Rubicon.
Mutations in the gene encoding the protein kinase CDKL5 cause a debilitating neurodevelopmental disease termed CDKL5 disorder. The impact of these mutations on CDKL5 function is poorly understood ...because the substrates and cellular processes controlled by CDKL5 are unclear. Here, we describe a quantitative phosphoproteomic screening which identified MAP1S, CEP131 and DLG5—regulators of microtubule and centrosome function—as cellular substrates of CDKL5. Antibodies against MAP1S phospho‐Ser900 and CEP131 phospho‐Ser35 confirmed CDKL5‐dependent phosphorylation of these targets in human cells. The phospho‐acceptor serine residues in MAP1S, CEP131 and DLG5 lie in the motif RPXSA, although CDKL5 can tolerate residues other than Ala immediately C‐terminal to the phospho‐acceptor serine. We provide insight into the control of CDKL5 activity and show that pathogenic mutations in CDKL5 cause a major reduction in CDKL5 activity in vitro and in cells. These data reveal the first cellular substrates of CDKL5, which may represent important biomarkers in the diagnosis and treatment of CDKL5 disorder, and illuminate the functions of this poorly characterized kinase.
Synopsis
CDKL5 kinase is mutated in a neurodevelopmental disease termed CDKL5 disorder but the cellular targets and functions of CDKL5 are unclear. A phosphoproteomic screen to identify cellular targets of CDKL5 now addresses this gap.
CDKL5 phosphorylates MAP1S, CEP131, and DLG5, proteins involved in regulation of microtubules and centrosomes.
In all three substrates, the phosphorylated serine lies in an RPXSA motif, with in vitro analysis showing certain amino acids other than Ala also tolerated C‐terminal to the phosphoacceptor serine.
CDKL5 activity is controlled by tyrosine auto‐phosphorylation in its T‐loop.
Pathogenic CDKL5 mutations cause severe reduction in kinase activity towards MAP1S and CEP131 in cells and in vitro.
Identification of phosphorylation targets reveals a sequence preference for the neurodevelopmental disease‐linked CDKL5, and that pathogenic mutations decrease activity towards microtubule‐ and centrosome‐associated substrates.
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