Altering the bilayer motif in ERBB2 HER2 TMD and in ErbB HER TMD dimer causing in vitro and in vivo tumor suppression

Human ERBB2 is a transmembrane signaling tyrosine kinase receptor, which seems an ideal target of human WNT16B, the secreted growth factor possibly causes transmembrane domain (TMD) mutations. There is a strong relationship between the chemical nature of the TMD mutations and the potency with which they activate HER2. In silico, we modeled the possible docking conformation of human WNT16B and human ERBB2 TMD homodimer, resulted a mutant complex. The ribbon structure, the C-terminal and N-terminal and GG4-like motif structures are similar in HER2 TMD and HER TMD, we modeled WNTl6B’s possible docking conformation to the HER1 TMD (ErbB), also resulted a mutant complex. If there is a strong relationship between TMD mutations improving the active dimer interface or stabilizing an activated conformation and the potency with which they activate HER2 (and possibly also HER), than the TMD dimerization part seems ideal reagent-target. The agent we tested – the 4-(Furan-2-yl)hepta-1,6-dien-4-ol (AKOS004122375) – has very good connectivity attributes by its several rotatable bonds, and according to the in silico inspection of close residues intermolecular bonds, and the ligand docking, it can straight connect to human ERBB2 TMD (HER2), and to the ErbB TMD (HER1) dimer bilayer motif as well. In silico, we also tested the agent ligand’s docking into the residues of human WNT16B and human ERBB2 TMD (HER2) mutant complex, and human WNT16B and human ErbB TMD (HER1) mutant complex. We tested the agent ligand in vitro and in vivo in several tumor models, highlighting that targeting the EGFR’s TMD with an agent not only reduces treatment-induced metastasis, but radically decreases the tumor growth as well. Because of the analogous structure of HER2 TMD and HER TMD, this dimerization motif-targeting can also be successful in HER and HER2 EGFR signaling. In vitro, we reached 80-94% proliferation percentage in different tumor models, in vivo we reached 35-61% tumor suppression in different tumor models, the metastasis inhibition effect of the compound was 82-87% in different tumor models.

Enhancement of the thermostability of mouse claudin-3 on complex formation with the carboxyl-terminal region ofClostridium perfringensenterotoxin improves crystal quality

Tight junctions regulate substance permeation through intercellular spaces as a physical barrier or a paracellular pathway, and play an important role in maintaining the internal environment. Claudins, which are tetraspan-transmembrane proteins, are pivotal components of tight junctions. In mammals 27 claudin subtypes have been identified, each of which interacts with specific subtypes. Although the crystal structures of several subtypes have been determined, the molecular mechanisms underlying subtype specificity remain unclear. Here, mouse claudin-3 (mCldn3) was crystallized in complex with the C-terminal region ofClostridium perfringensenterotoxin (C-CPE) for the structural analysis of an additional claudin subtype. mCldn3 alone was difficult to crystallize, but complex formation with C-CPE enhanced the thermostability of mCldn3 and facilitated its crystallization. The introduction of an S313A mutation into C-CPE further improved its thermostability, and the resolution limits of the diffraction data sets improved from 8 Å for the wild-type complex to 4.7 Å for the S313A mutant complex.

The CUL3–KLHL3 E3 ligase complex mutated in Gordon's hypertension syndrome interacts with and ubiquitylates WNK isoforms: disease-causing mutations in KLHL3 and WNK4 disrupt interaction

The WNK (with no lysine kinase)–SPAK (SPS1-related proline/alanine-rich kinase)/OSR1 (oxidative stress-responsive kinase 1) signalling pathway plays an important role in controlling mammalian blood pressure by modulating the activity of ion co-transporters in the kidney. Recent studies have identified Gordon's hypertension syndrome patients with mutations in either CUL3 (Cullin-3) or the BTB protein KLHL3 (Kelch-like 3). CUL3 assembles with BTB proteins to form Cullin–RING E3 ubiquitin ligase complexes. To explore how a CUL3–KLHL3 complex might operate, we immunoprecipitated KLHL3 and found that it associated strongly with WNK isoforms and CUL3, but not with other components of the pathway [SPAK/OSR1 or NCC (Na+/Cl− co-transporter)/NKCC1 (Na+/K+/2Cl− co-transporter 1)]. Strikingly, 13 out of the 15 dominant KLHL3 disease mutations analysed inhibited binding to WNK1 or CUL3. The recombinant wild-type CUL3–KLHL3 E3 ligase complex, but not a disease-causing CUL3–KLHL3[R528H] mutant complex, ubiquitylated WNK1 in vitro. Moreover, siRNA (small interfering RNA)-mediated knockdown of CUL3 increased WNK1 protein levels and kinase activity in HeLa cells. We mapped the KLHL3 interaction site in WNK1 to a non-catalytic region (residues 479–667). Interestingly, the equivalent region in WNK4 encompasses residues that are mutated in Gordon's syndrome patients. Strikingly, we found that the Gordon's disease-causing WNK4[E562K] and WNK4[Q565E] mutations, as well as the equivalent mutation in the WNK1[479–667] fragment, abolished the ability to interact with KLHL3. These results suggest that the CUL3–KLHL3 E3 ligase complex regulates blood pressure via its ability to interact with and ubiquitylate WNK isoforms. The findings of the present study also emphasize that the missense mutations in WNK4 that cause Gordon's syndrome strongly inhibit interaction with KLHL3. This could elevate blood pressure by increasing the expression of WNK4 thereby stimulating inappropriate salt retention in the kidney by promoting activation of the NCC/NKCC2 ion co-transporters. The present study reveals how mutations that disrupt the ability of an E3 ligase to interact with and ubiquitylate a critical cellular substrate such as WNK isoforms can trigger a chronic disease such as hypertension.

The regulation of AMP-activated protein kinase by phosphorylation

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.