Characterization of acrosin and acrosin binding protein as novel CRISP2 interacting proteins in boar spermatozoa.

BACKGROUND: Previously, we reported that cysteine-rich secretory protein 2 is involved in high molecular weight complexes in boar spermatozoa. These cysteine-rich secretory protein 2protein complexes are formed at the last phase of sperm formation in the testis and play a role in sperm shaping and functioning. OBJECTIVES: This study aimed to identify cysteine-rich secretory protein 2 interacting partners. These binding partner interactions were investigated under different conditions, namely, non-capacitating conditions, after the induction of in vitro sperm capacitation and subsequently during an ionophore A23187-induced acrosome reaction. MATERIALS AND METHODS: The incubated pig sperm samples were subjected to protein extraction. Extracted proteins were subjected to blue native gel electrophoresis and native immunoblots. Immunoreactive gel bands were excised and subjected to liquid chromatography-mass spectrometry (LC-MS) analysis for protein identification. Protein extracts were also subjected to CRISP2 immunoprecipitation and analyzed by LC-MS for protein identification. The most prominent cystein-rich secretory protein 2 interacting proteins that appeared in both independent LC-MS analyses were studied with a functional in situ proximity interaction assay to validate their property to interact with cystein-rich secretory protein 2 in pig sperm. RESULTS: Blue native gel electrophoresis and native immunoblots revealed that cystein-rich secretory protein 2 was present within a ∼150 kDa protein complex under all three conditions. Interrogation of cystein-rich secretory-protein 2-immunoreactive bands from blue native gels as well as cystein-rich secretory protein 2 immunoprecipitated products using mass spectrometry consistently revealed that, beyond cystein-rich secretory protein 2, acrosin and acrosin binding protein were among the most abundant interacting proteins and did interact under all three conditions. Co-immunoprecipitation and immunoblotting indicated that cystein-rich secretory protein 2 interacted with pro-acrosin (∼53 kDa) and Aacrosin binding protein under all three conditions and additionally to acrosin (∼35 kDa) after capacitation and the acrosome reaction. The colocalization of these interacting proteins with cystein-rich secretory protein 2 was assessed via in situ proximity ligation assays. The colocalization signal of cystein-rich secretory protein 2 and acrosin in the acrosome seemed dispersed after capacitation but was consistently present in the sperm tail under all conditions. The fluorescent foci of cystein-rich secretory protein 2 and acrsin binding protein colocalization appeared to be redistributed within the sperm head from the anterior acrosome to the post-acrosomal sheath region upon capacitation. DISCUSSION AND CONCLUSION: These results suggest that CRISP2 may act as a scaffold for protein complex formation and dissociation to ensure the correct positioning of proteins required for the acrosome reaction and zona pellucida penetration.

Fasting improves therapeutic response in hepatocellular carcinoma through p53-dependent metabolic synergism.

Cancer cells voraciously consume nutrients to support their growth, exposing metabolic vulnerabilities that can be therapeutically exploited. Here, we show in hepatocellular carcinoma (HCC) cells, xenografts, and patient-derived organoids that fasting improves sorafenib efficacy and acts synergistically to sensitize sorafenib-resistant HCC. Mechanistically, sorafenib acts noncanonically as an inhibitor of mitochondrial respiration, causing resistant cells to depend on glycolysis for survival. Fasting, through reduction in glucose and impeded AKT/mTOR signaling, prevents this Warburg shift. Regulating glucose transporter and proapoptotic protein expression, p53 is necessary and sufficient for the sorafenib-sensitizing effect of fasting. p53 is also crucial for fasting-mediated improvement of sorafenib efficacy in an orthotopic HCC mouse model. Together, our data suggest fasting and sorafenib as rational combination therapy for HCC with intact p53 signaling. As HCC therapy is currently severely limited by resistance, these results should instigate clinical studies aimed at improving therapy response in advanced-stage HCC.

Influence of dynamic flow environment on nanoparticle-protein corona: From protein patterns to uptake in cancer cells.

The fast growing use of nanoparticles (NPs) in biotechnology and biomedicine raises concerns about human health and the environment. When introduced in physiological milieus, NPs adsorb biomolecules (especially proteins) forming the so-called protein corona (PC). As it is the PC that mostly interacts with biological systems, it represents a major element of the NPs' biological identity with impact on nanotoxicology, nanosafety and targeted delivery of nanomedicines. To date, NP-protein interactions have been largely investigated in vitro, but this condition is far from mimicking the dynamic nature of physiological environments. Here we investigate the effect of shear stress on PC by exposing lipid NPs with different surface chemistry (either unmodified and PEGylated) to circulating fetal bovine serum (FBS). PC formed upon in vitro incubation was used as a reference. We demonstrate that PC is significantly influenced by exposure to dynamic flow and that changes in PC composition are dependent on both exposure time and NP's surface chemistry. Notably, alterations induced by dynamic flow affected cellular uptake of lipid NPs in both human cervical cancer (HeLa) and human breast adenocarcinoma (MCF7) cell lines.