Search results
Found 2400 matches for
A new paradigm of islet adaptations in human pregnancy: insights from immunohistochemistry and proteomics
Abstract Physiological changes during pregnancy support foetal growth, including adaptations in pancreatic islets to maintain glucose homeostasis. We investigate these adaptations using rare, high-quality pancreatic tissue from pregnant human donors and matched controls. We profile islets from pregnant donors using proteomics and assess α- and β-cell characteristics, as well as prolactin receptor and serotonin 2B receptor expression. Proteomic profiling of microdissected human islets identifies 7546 proteins but shows minimal differences in protein expression. In pregnancy, we show that islet area increases 1.9-fold, α- and β-cell areas increase 4.3- and 1.9-fold, driven by an increase in cell number rather than hypertrophy. Prolactin receptor expression is higher in α but not β cells, and serotonin 2B receptor is undetectable in β cells. Glucagon-like peptide-1 abundance increases 2.9-fold in α cells. These findings indicate that the molecular mechanisms driving pregnancy-induced islet adaptations in humans differ from those in mice, highlighting the need for human-based studies.
Amyloid-β disrupts APP-regulated protein aggregation and dissociation from recycling endosomal membranes.
Secretory proteins aggregate into non-soluble dense-core granules in recycling endosome-like compartments prior to regulated release. By contrast, aberrantly processed, secreted amyloid-β (Aβ) peptides derived from amyloid precursor protein (APP) form pathological extracellular amyloidogenic aggregations in late-stage Alzheimer's disease (AD). By examining living Drosophila prostate-like secondary cells, we show that both APP and Aβ peptides affect normal biogenesis of dense-core granules. These cells generate dense-core granules and secreted nanovesicles called Rab11-exosomes via evolutionarily conserved mechanisms within highly enlarged secretory compartments with recycling endosomal identity. The fly APP homologue, APP-like (APPL), associates with these vesicles and the compartmental limiting membrane, from where its extracellular domain modulates protein aggregation. Proteolytic release of this domain permits mini-aggregates to coalesce into a large central dense-core granule. Mutant Aβ expression disrupts this process and compartment motility, and increases aberrant lysosomal targeting, mirroring previously unexplained early-stage pathological events in AD. It also promotes cell-to-cell propagation of these endolysosomal defects, again phenocopying changes observed in AD. Our data therefore demonstrate physiological roles for APP in membrane-dependent protein aggregation, involving molecular mechanisms, which when disrupted by Aβ peptides, trigger Alzheimer's disease-relevant pathologies.
The Src family kinase inhibitor drug Dasatinib and glucocorticoids display synergistic activity against tongue squamous cell carcinoma and reduce MET kinase activity
Background: Tongue squamous cell carcinoma (TSCC) is an aggressive cancer associated with a poor prognosis and limited treatment options, necessitating new drug targets to improve therapeutic outcomes. Our current work studies protein tyrosine kinases as well-known targets for successful cancer therapies. It focuses on Src family kinases (SFK), which are known to play a critical role in some head and neck tumors. Methods: Western blot analyses of phospho-tyrosine protein patterns in 34 TSCC lines facilitated the investigation of SFK as contributors to these phosphorylations. The SFK inhibitors PP2 and Dasatinib were utilized to determine SFK contributions to cell motility and survival. A high-throughput screen with 1600 FDA-approved drugs was performed with three TSCC lines to discover drugs that act synergistically with Dasatinib against TSCC cell viability. Glucocorticoids emerged as potential candidates and were further investigated in 2D culture and by 3D soft agar colony formation. Dexamethasone was chosen as the major tool for our analyses of synergistic effects of Dasatinib and glucocorticoids on TSCC lines. Effects on the cell cycle were investigated by flow cytometry and expression levels of cell cycle regulators. Senescence was analyzed by senescence-associated β galactosidase detection and p27Kip1 protein expression. Autophagy was measured by Acridine Orange staining. Results: A panel of 34 TSCC lines showed a surprisingly homogenous pTyr-protein pattern and a prominent 130 kDa pTyr-protein. Inhibition of SFK activity greatly reduced overall pTyr-protein levels and p130Cas tyrosine phosphorylation. It also impaired TSCC viability in 2D cell culture and 3D soft agar colony formation. A high-throughput drug combination screen with Dasatinib identified glucocorticoids as promising candidates for synergistic activity. Dasatinib and Dexamethasone combination treatment showed strong synergistic effects on Src and p130Cas phosphorylation and led to reduced p130Cas expression. Dexamethasone also suppressed phosphorylation of the MET kinase and its key substrate Gab1. On the cellular level, Dasatinib combination with glucocorticoids led to G1 cell cycle arrest, appeared to increase senescence and enhanced autophagy. This was also reflected by effects on cell cycle regulatory proteins, including CDKs and cyclins. Conclusion: This work is the first to show a strong synergistic activity of Dasatinib in combination with clinically used glucocorticoids in solid tumors. Furthermore, the tyrosine kinase MET and its effector protein Gab1 are newly identified glucocorticoid targets. Given the extensive research on MET as a drug target in various cancers, our findings have the potential to advance future cancer treatments.
Acidosis attenuates the hypoxic stabilization of HIF-1α by activating lysosomal degradation
Hypoxia-inducible factors (HIFs) mediate cellular responses to low oxygen, notably enhanced fermentation that acidifies poorly perfused tissues and may eventually become more damaging than adaptive. How pH feeds back on hypoxic signaling is unclear but critical to investigate because acidosis and hypoxia are mechanistically coupled in diffusion-limited settings, such as tumors. Here, we examined the pH sensitivity of hypoxic signaling in colorectal cancer cells that can survive acidosis. HIF-1α stabilization under acidotic hypoxia was transient, declining over 48 h. Proteomic analyses identified responses that followed HIF-1α, including canonical HIF targets (e.g., CA9, PDK1), but these did not reflect a proteome-wide downregulation. Enrichment analyses suggested a role for lysosomal degradation. Indeed, HIF-1α destabilization was blocked by inactivating lysosomes, but not proteasome inhibitors. Acidotic hypoxia stimulated lysosomal activity and autophagy via mammalian target of rapamycin complex I (mTORC1), resulting in HIF-1α degradation. This response protects cells from excessive acidification by unchecked fermentation. Thus, alkaline conditions are permissive for at least some aspects of HIF-1α signaling.
Activity-Based Protein Profiling (ABPP) of Cellular DeISGylating Enzymes and Inhibitor Screening.
A detailed methodology platform is described for activity-based protein profiling (ABPP) of cellular deISGylating enzymes using a specific activity-based interferon-stimulated gene 15 (ISG15) probe. Manual and semi-automated workflows for medium- to high-throughput applications are outlined in this chapter, with western blotting and proteomics-based techniques as the main readouts. This methodology informs us of endogenous deISGylating enzyme expression and activity in a cellular context, including USP18, the type I interferon (IFN-I)-inducible deISGylase, and several constitutively expressed deubiquitinases (DUBs), such as USP5, USP14, USP16, and USP36, that exert cross-reactivity to ISG15. ISG15-ABPP also enables the identification and characterization of potent and selective deISGylating enzyme modulators.
Characterization of ADAMTS9 proteoglycanase activity: comparison with ADAMTS1, ADAMTS4 and ADAMTS5.
A Disintegrin-like And Metalloprotease domain with Thrombospondin type I motifs (ADAMTS) 9 has essential, non-redundant roles during embryogenesis. Adamts9 null murine embryos die prior to completing gastrulation. Unusually for a protease, Adamts9 haploinsufficiency results in cardiovascular and ocular anomalies. ADAMTS9 is required for proteostasis of versican, a widely distributed large aggregating proteoglycan abundant in the provisional extracellular matrix during embryogenesis. Despite its importance, ADAMTS9 proteoglycanase activity has undergone limited characterization, especially in comparison to ADAMTS1, ADAMTS4, and ADAMTS5, due to difficulties in expressing and purifying the >200 kDa full-length form of ADAMTS9. Like ADAMTS1, ADAMTS4, and ADAMTS5, ADAMTS9 cleaves versican V1 isoform at E441-A442, but unlike them, cleavages at other sites are unknown. Here, we expressed a truncated ADAMTS9 construct (ADAMTS9 MDTCS) consisting of all ADAMTS 'core domains' present in ADAMTS1, ADAMTS4, and ADAMTS5, and characterized its activity against versican, aggrecan, and the small leucine-rich proteoglycan biglycan. We identified cleavages in versican (V1 and V2 isoforms) and biglycan using a z-score approach based on label-free quantitation of semi- and fully tryptic/GluC peptides. Moreover, using a quantitative assay, we established that ADAMTS9 MDTCS versicanase activity at the E441-A442 site is 175-fold lower than ADAMTS5, 9-fold lower than ADAMTS4, and 5.5-fold higher than ADAMTS1. Finally, we confirmed that ADAMTS9 MDTCS cleaves bovine aggrecan at E392-A393. This analysis of the proteoglycanase activity in the ADAMTS family highlights differences and similarities in cleavage site specificities which could be leveraged to develop selective small molecule inhibitors against current targets of interest, ADAMTS4, ADAMTS5, and ADAMTS7.
Deep palaeoproteomic profiling of archaeological human brains.
Palaeoproteomics leverages the persistence, diversity, and biological import of ancient proteins to explore the past, and answer fundamental questions about phylogeny, environment, diet, and disease. These insights are largely gleaned from hard tissues like bone and teeth, as well-established protocols exist for extracting ancient proteins from mineralised tissues. No such method, however, exists for the soft tissues, which are underexplored in palaeoproteomics given permission for destructive analysis routinely depends on a proven methodology. Considering less than one-tenth of all human proteins are expressed in bone, compared to three-quarters in the internal organs, the amount of biological information presently inaccessible is substantial. We address this omission with an optimised LC-FAIMS-MS/MS workflow yielding the largest, most diverse palaeoproteome yet described. Using archaeological human brains, we test ten protocols with varied chemistries and find that urea lysis effectively disrupts preserved membrane regions to expose low-abundant, intracellular analytes. Further, we show that ion mobility spectrometry improves unique protein identification by as much as 40%, and represents a means of "cleaning" dirty archaeological samples. Our methodology will be useful for improving protein recovery from a range of ancient tissues and depositional environments.
Effect of a Laparoscopic Donor Nephrectomy in Healthy Living Kidney Donors on the Acute Phase Response Using Either Propofol or Sevoflurane Anesthesia
Surgical trauma elicits a complex inflammatory stress response, contributing to postoperative morbidity and recovery variability. This response is influenced by patient-specific factors and surgical and anesthetic techniques. To isolate the impact of anesthesia on the acute phase response, we investigated plasma proteomic changes in a uniquely homogeneous cohort of healthy, living kidney donors (n = 36; propofol = 19; sevoflurane = 17) undergoing laparoscopic donor nephrectomy. Proteomic profiling of plasma samples collected preoperatively and at 2 and 24 h postoperatively revealed 633 quantifiable proteins, of which 22 showed significant perioperative expression changes. Eight proteins exhibited over two-fold increases, primarily related to the acute phase response (CRP, SAA1, SAA2, LBP), tissue repair (FGL1, A2GL), and anti-inflammatory regulation (AACT). These changes were largely independent of anesthetic type, though SAA2 and MAN1A1 showed anesthetic-specific expression. The upregulation of these proteins implicates the activation of immune pathways involved in host defense, tissue remodeling, and inflammation resolution. Our findings provide a molecular reference for the surgical stress response in healthy individuals and highlight candidate biomarkers for predicting and managing postoperative outcomes. Understanding these pathways may support the development of strategies to mitigate surgical stress and enhance recovery, particularly in vulnerable patient populations.
N-terminal cysteine acetylation and oxidation patterns may define protein stability
AbstractOxygen homeostasis is maintained in plants and animals by O2-sensing enzymes initiating adaptive responses to low O2 (hypoxia). Recently, the O2-sensitive enzyme ADO was shown to initiate degradation of target proteins RGS4/5 and IL32 via the Cysteine/Arginine N-degron pathway. ADO functions by catalysing oxidation of N-terminal cysteine residues, but despite multiple proteins in the human proteome having an N-terminal cysteine, other endogenous ADO substrates have not yet been identified. This could be because alternative modifications of N-terminal cysteine residues, including acetylation, prevent ADO-catalysed oxidation. Here we investigate the relationship between ADO-catalysed oxidation and NatA-catalysed acetylation of a broad range of protein sequences with N-terminal cysteines. We present evidence that human NatA catalyses N-terminal cysteine acetylation in vitro and in vivo. We then show that sequences downstream of the N-terminal cysteine dictate whether this residue is oxidised or acetylated, with ADO preferring basic and aromatic amino acids and NatA preferring acidic or polar residues. In vitro, the two modifications appear to be mutually exclusive, suggesting that distinct pools of N-terminal cysteine proteins may be acetylated or oxidised. These results reveal the sequence determinants that contribute to N-terminal cysteine protein modifications, with implications for O2-dependent protein stability and the hypoxic response.