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Cell-surface ancillary glycoproteins basigin or embigin form heterodimeric complexes with proton-coupled monocarboxylate transporters (MCTs), facilitating the membrane trafficking of MCTs and regulating their transport activities. Here, we determine the cryoelectron microscopy (cryo-EM) structure of the human MCT1-embigin complex and observe that embigin forms extensive interactions with MCT1 to facilitate its localization to the plasma membrane. In addition, the formation of the heterodimer effectively blocks MCT1 from forming a homodimer through a steric hindrance effect, releasing the coupling between two signature motifs and driving a significant conformation change in transmembrane helix 5 (TM5) of MCTs. Consequently, the substrate-binding pocket alternates between states of homodimeric coupling and heterodimeric decoupling states and exhibits differences in substrate-binding affinity, supporting the hypothesis that the substrate-induced motion originating in one subunit of the MCT dimer could be transmitted to the adjacent subunit to alter its substrate-binding affinity.

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. The current study aimed to elucidate the mechanism by which exosomes carrying KRAS mutant contribute to neutrophil recruitment as well as the formation of the neutrophil extracellular traps (NETs) in CRC. Methods: APC-WT and APC-KRASG12D mouse models were initially established. The peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Next, exosomes isolated from APC-WT and APC-KRASG12D mice were injected into APC-WT and APC-KRASG12D mice. The neutrophil ratio, NETs formation and IL-8 protein content in colon tissue were subsequently quantified. DKs-8 (wild type) and DKO-1 (KRAS mutant) cells were employed for in vitro experimentation. Then, DKs-8 cells were cultured with exosome-treated PMA stimulated neutrophil-forming NETs culture medium, with cell viability, invasion, migration, and adhesion evaluated. Results: Compared with APC-WT mice, the number of polyps and neutrophils in the peripheral blood, spleen and mLNs increased in APC-KRASG12D mice, with increased NETs formation, IL-8 expression, and exosomes. IL-8 upregulation, neutrophil recruitment and NETs formation were observed in mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed by DKO-1-Exosomes, which were inhibited by DNAse. In addition, DKs-8 and DKO-1 cells-derived exosomes can adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert a stimulatory effect on the production of IL-8 while promoting NETs formation as well as CRC promotion. Conclusion: The results obtained provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger an increase in IL-8 production, promote neutrophil recruitment and form NETs, ultimately leading to the promotion of CRC. Funding Statement: This work was supported by the National Natural Science Foundation of China (81873975, 81802084, 81974314, 81902984), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Medical Guidace Science and Technology Support Project of Shanghai (19411964800), the Natural Science Foundation of Shanghai (19ZR1448800), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital [ITJ(ZD)1803, ITJ(ZD)1905, ITJ(QN)1905]. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the Ethics Committee of Tongji Hospital of Tongji University. All subjects have written informed consent. All animal use and experiments were performed in strict accordance with the procedures approved by the National Cancer Institute Animal Care and Use Committee (ACUC).

Mechanisms of angiotensin II and cAMP regulating the expression of angiotensin II type 1 (AT(1)) receptor mRNA were studied in neonatal rat cardiomyocytes. Angiotensin II induced a transient decrease of AT(1)-mRNA expression in time- and dose-dependent manner. Maximal decrease (49.2 +/- 9.5% of control) occurred at 6 h of angiotensin II (10 nmol/l) treatment. AT(1) receptor antagonists 4-ethyl-2-n-propyl-1-[2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole-5-carboxylic acid (DMP811) and losartan as well as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) reversed the down-regulation of AT(1)-mRNA expression. 6 h of phorbol 12-myristate 13-acetate (PMA) stimulation caused a decrease of AT(1)-mRNA level. Treatment by angiotensin II plus actinomycin D for 6 h produced the same effect as actinomycin D alone. These results suggest that angiotensin II down-regulates AT(1)-mRNA level of rat cardiomyocytes by inhibiting the transcription of AT(1) gene, which is mediated by AT(1) receptor and related to the activation of protein kinase C. Stimulation by forskolin plus 3-isobutyl-1-methyl-xanthine (IBMX) decreased the expression of AT(1)-mRNA to 68.1 +/- 21.5% of control at 6 h treatment; while increased to 207.9 +/- 27.1% of control at 48 h treatment. A series of 5'-upstream deletion mutants of AT(1A) promoter were produced and then were recombined with pGL(3) basic vector utilizing luciferase as reporter gene. Among all the constructors, p(-201/+ 74)Luc was of the highest luciferase activity (5.9 times higher than control) after stimulation by forskolin for 48 h. Further deletion from -201 to -61 resulted in a large decrease of activity. These results indicate that cAMP induces a time-dependent bi-directional regulation of AT(1)-mRNA expression. The cAMP responsible element (CRE) cis-element located in the region -201/-61 of rat AT(1A) promoter is forskolin inducible, which may mediate the up-regulation of AT(1)-mRNA expression induced by cAMP long-lasting stimulation.