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Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation -- whose levels and intracellular localization are regulated by Munc18-2 -- remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion. [ABSTRACT FROM AUTHOR]

Keywords: target identification; photoaffinity labelling; fluorescence probes; proteomics; proteins The labelling strategy enabled successful photo-crosslinking with target proteins in both insect cell lysates and mammalian live cells, demonstrating the efficient and selective labelling of Src. Target identification, photoaffinity labelling, fluorescence probes, proteomics, proteins. [Extracted from the article]

Background: Both the type I insulin-like growth factor receptor (IGF1R) and Src pathways are associated with the development and progression of numerous types of human cancer, and Src activation confers resistance to anti-IGF1R therapies. Hence, targeting both IGF1R and Src concurrently is one of the main challenges in combating resistance to the currently available anti-IGF1R-based anticancer therapies. However, the enhanced toxicity from this combinatorial treatment could be one of the main hurdles for this strategy, suggesting the necessity of developing a novel strategy for co-targeting IGF1R and Src to meet an urgent clinical need. Methods: We synthesized a series of 4-aminopyrazolo[3,4-d]pyrimidine-based dual IGF1R/Src inhibitors, selected LL28 as an active compound and evaluated its potential antitumor effects in vitro and in vivo using the MTT assay, colony formation assays, flow cytometric analysis, a tumor xenograft model, and the KrasG12D/+-driven spontaneous lung tumorigenesis model. Results: LL28 markedly suppressed the activation of IGF1R and Src and significantly inhibited the viability of several NSCLC cell lines in vitro by inducing apoptosis. Administration of mice with LL28 significantly suppressed the growth of H1299 NSCLC xenograft tumors without overt toxicity and substantially reduced the multiplicity, volume, and load of lung tumors in the KrasG12D/+-driven lung tumorigenesis model. Conclusions: The present results suggest the potential of LL28 as a novel anticancer drug candidate targeting both IGF1R and Src, providing a new avenue to efficient anticancer therapies. Further investigation is warranted in advanced preclinical and clinical settings. [ABSTRACT FROM AUTHOR]

Background: Trunk co-contraction during lifting may reflect a guarded motor response to a threatening task. This work estimated the impact of pain catastrophizing on trunk co-contraction during lifting, in people with and without low back pain., Methods: Adults with high pain catastrophizing (back pain: n = 29, healthy: n = 7) and low pain catastrophizing (back pain: n = 20, healthy: n = 11), performed 10 repetitions of a lifting task. Electromyography data of rectus abdominis, erector spinae and external oblique muscles were collected, bilaterally. Co-contraction indices were determined for rectus abdominis/erector spinae and external oblique/erector spinae pairings, bilaterally. Pain catastrophizing was measured using the pain catastrophizing scale and task-specific fear using the Photograph series of daily activities scale. Three-way mixed ANOVAs tested the effects of group (back pain vs. healthy), pain catastrophizing (high vs. low), lifting phase (lifting vs. replacing) and their interactions., Results: There were no main effects of pain catastrophizing, lifting phase, nor any interactions (p > 0.05). Group effects revealed greater co-contraction for bilateral erector spinae/rectus abdominis pairings (but not erector spinae-external oblique pairings) in people with back pain, compared to healthy participants, independent of pain catastrophizing and lifting phase (p < 0.05). Spearman correlations associated greater task-specific fear and greater erector spinae-left external oblique co-contraction, only in people with back pain (p < 0.05)., Conclusions: Greater co-contraction in the back pain group occurred independent of pain catastrophizing, as measured with a general questionnaire. A task-specific measure of threat may be more sensitive to detecting relationships between threat and co-contraction., Significance Statement: This work contributes evidence that people with back pain commonly exhibit trunk co-contraction when lifting. The lack of a relationship between pain catastrophizing and trunk co-contraction, however, challenges evidence linking psychological factors and guarded motor behaviour in this group. Together, this suggests that other factors may be stronger determinants of co-contraction in people with LBP or that a general construct like pain catastrophizing may not accurately represent this relationship., (© 2024 The Author(s). European Journal of Pain published by John Wiley & Sons Ltd on behalf of European Pain Federation ‐ EFIC ®.)

Rationale: The type I insulin-like growth factor receptor (IGF-1R) signaling pathway plays key roles in the development and progression of numerous types of human cancers, and Src and AXL have been found to confer resistance to anti-IGF-1R therapies. Hence, co-targeting Src and AXL may be an effective strategy to overcome resistance to anti-IGF-1R therapies. However, pharmacologic targeting of these three kinases may result in enhanced toxicity. Therefore, the development of novel multitarget anticancer drugs that block IGF-1R, Src, and AXL is urgently needed. Methods: We synthesized a series of phenylpyrazolo[3,4- d ]pyrimidine (PP)-based compounds, wherein the PP module was conjugated with 2,4-bis-arylamino-1,3-pyrimidines (I2) via a copper(I)-catalyzed alkyne-azide cycloaddition reaction. To develop IGF-1R/Src/AXL-targeting small molecule kinase inhibitors, we selected LL6 as an active compound and evaluated its antitumor and antimetastatic effects in vitro and in vivo using the MTT assay, colony formation assays, migration assay, flow cytometric analysis, a tumor xenograft model, the Kras G12D/+ -driven spontaneous lung tumorigenesis model, and a spontaneous metastasis model using Lewis lung carcinoma (LLC) allografts. We also determined the toxicity of LL6 in vitro and in vivo . Results: LL6 induced apoptosis and suppressed viability and colony-forming capacities of various non-small cell lung cancer (NSCLC) cell lines and their sublines with drug resistance. LL6 also suppressed the migration of NSCLC cells at nontoxic doses. Administration of LL6 in mice significantly suppressed the growth of NSCLC xenograft tumors and metastasis of LLC allograft tumors with outstanding toxicity profiles. Furthermore, the multiplicity, volume, and load of lung tumors in Kras G12D/+ transgenic mice were substantially reduced by the LL6 treatment. Conclusions: Our results show the potential of LL6 as a novel IGF-1R/Src/AXL-targeting small molecule kinase inhibitor, providing a new avenue for anticancer therapies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)