Molecular conversion of MIG6 hotspot-3 peptide from the nonbinder to a moderate binder of HER2 by rational design of an orthogonal interaction system at the HER2-peptide interface.

Human epidermal growth factor receptor 2 (HER2) has been established as an approved druggable target for the treatment of patients with diverse gynecological tumors such as ovarian, cervical and breast cancers. The mitogen-inducible gene 6 (MIG6) protein is a negative regulator of HER2 signaling by using its Seg1 segment to disrupt the allosteric dimerization of HER2 kinase domain. Previous studies found that the Seg1 adopts three separated hotspots to interact with the HER2 dimerization interface, in which the third hotspot (H3) is located at the core region of the interface but its derived H3 peptide (356PKYVS360) and Tyr358Phe mutant (356PKFVS360) cannot bind effectively to the interface in an independent manner. In this study, we demonstrate that the H3 peptide can be converted from nonbinder to a moderate binder of HER2 by just adding an orthogonal noncovalent interaction system (X⋯O┄H) between a halogen bond (X⋯O) and a hydrogen bond (H┄O) involving peptide Phe358 residue and HER2 Val948/Trp951 residues. High-level calculations are utilized to rigorously characterize and rationally design the X⋯O┄H system, which is then optimized with different halogen atoms and at different substituting positions. It is revealed that there is a synergistic effect between the X⋯O and H┄O of the orthogonal interaction system; formation of the halogen bond can enhance the interaction strength of the hydrogen bond. In silico analysis and in vitro assay reach a consistence that Br-substitution at the m-position of peptide Phe358 phenyl moiety is the best choice that can render strong interaction for the X⋯O┄H system, which also makes the peptide 'bindable' to HER2 kinase domain, while F/Cl/I-substitution at the same position can only improve the peptide affinity moderately or modestly. In contrast, the Br-substitution at the o- and p-positions of peptide Phe358 phenyl moiety cannot define effective X⋯O┄H interaction and thus does not confer additional affinity to the HER2-peptide complex.

Revisiting bone morphogenetic protein-2 knuckle epitope and redesigning the epitope-derived peptides.

The bone morphogenetic protein-2 (BMP2) plays a crucial role in bone formation, growth and regeneration, which adopts a conformational wrist epitope and a linear knuckle epitope to interact with its type-I (BRI) and type-II (BRII) receptors, respectively. In this study, we systematically examine the BRII-recognition site of BMP2 at structural, energetic and dynamic levels and accurately locate hotspots of the recognition at BMP2-BRII complex interface. It is revealed that the traditional knuckle epitope (BMP2 residue range 73-92) do fully match the identified hotspots; the BMP2-recognition site includes the C-terminal region of traditional knuckle epitope as well as its flanked ß-strands. In addition, the protein context of full-length BMP2 is also responsible for the recognition by addressing conformational constraint on the native epitope segment. Therefore, we herein redefine the knuckle epitope to BMP2 residue range 84-102, which has a similar sequence length but is slid along the protein sequence by ~10 residues as compared to traditional knuckle epitope. The redefined one is also a linear epitope that is natively a double-stranded ß-sheet with two asymmetric arms as compared to the natively single ß-strand of the traditional version, although their sequences are partially overlapped to each other. It is revealed that the redefined epitope-derived peptide LN84-102 exhibits an improved affinity by >3-fold relative to the traditional epitope-derived peptide KL73-92 . Even so, the LN84-102 peptide still cannot fully represent the BMP2 recognition event by BRII that has been reported to have a nanomolar affinity. We further introduce a disulfide bond across the two arms of double-stranded ß-sheet to constrain the free LN84-102 peptide conformation, which mimics the conformational constraint addressed by protein context. Consequently, several cyclic peptides are redesigned, in which the LN84-102 (cyc89-101) is determined to exhibit a sub-micromolar affinity; this value is ~5-fold higher than its linear counterpart. Structural analysis also reveals that the cyclic peptide can interact with BRII in a similar binding mode with the redefined knuckle epitope region in full-length BMP2 protein.

Downregulation of CCR5 inhibits the proliferation and invasion of cervical cancer cells and is regulated by microRNA-107.

Cervical cancer is among the most prevalent forms of cancer worldwide. C-C chemokine receptor type 5 (CCR5) is hypothesized to be a key functional protein involved in tumorigenesis. However, the role of CCR5 in cervical cancer remains unclear. Reverse transcription-quantitative polymerase chain reaction and western blot analysis were used to evaluate the mRNA and protein expression levels of CCR5 in human cervical carcinoma tissues. Furthermore, a small interfering RNA was employed to knockdown CCR5 in HeLa and C33A cells. MTT, colony formation and Transwell assays were performed to determine the effects of this knockdown on cell viability, proliferation and invasion. In addition, micro RNA (miR)-107 was identified as a potential candidate regulator of CCR5 using miR prediction algorithms, and the effects of miR-107 and its antisense miR on CCR5 mRNA expression were determined. The results of the present study indicated that CCR5 is overexpressed in human cervical cancer tissues compared with adjacent normal tissues, and its downregulation inhibits cervical cancer cell growth and proliferation. Furthermore, the downregulation of CCR5 appears to suppress cervical cancer cell invasion. Finally, the tumor suppressor miR-107 was able to directly target CCR5 and inhibit its expression. These results suggest that the upregulation of CCR5, which is inhibited by miR-107, may play a carcinogenic role in cervical cancer and could provide a novel therapeutic target in the future.