Promiscuous G-protein activation by the calcium-sensing receptor.

The human calcium-sensing receptor (CaSR) detects fluctuations in the extracellular Ca2+ concentration and maintains Ca2+ homeostasis1,2. It also mediates diverse cellular processes not associated with Ca2+ balance3-5. The functional pleiotropy of CaSR arises in part from its ability to signal through several G-protein subtypes6. We determined structures of CaSR in complex with G proteins from three different subfamilies: Gq, Gi and Gs. We found that the homodimeric CaSR of each complex couples to a single G protein through a common mode. This involves the C-terminal helix of each Gα subunit binding to a shallow pocket that is formed in one CaSR subunit by all three intracellular loops (ICL1-ICL3), an extended transmembrane helix 3 and an ordered C-terminal region. G-protein binding expands the transmembrane dimer interface, which is further stabilized by phospholipid. The restraint imposed by the receptor dimer, in combination with ICL2, enables G-protein activation by facilitating conformational transition of Gα. We identified a single Gα residue that determines Gq and Gs versus Gi selectivity. The length and flexibility of ICL2 allows CaSR to bind all three Gα subtypes, thereby conferring capacity for promiscuous G-protein coupling.

Structures revealing mechanisms of resistance and collateral sensitivity of Plasmodium falciparum to proteasome inhibitors.

The proteasome of the malaria parasite Plasmodium falciparum (Pf20S) is an advantageous drug target because its inhibition kills P. falciparum in multiple stages of its life cycle and synergizes with artemisinins. We recently developed a macrocyclic peptide, TDI-8304, that is highly selective for Pf20S over human proteasomes and is potent in vitro and in vivo against P. falciparum. A mutation in the Pf20S ß6 subunit, A117D, confers resistance to TDI-8304, yet enhances both enzyme inhibition and anti-parasite activity of a tripeptide vinyl sulfone ß2 inhibitor, WLW-vs. Here we present the high-resolution cryo-EM structures of Pf20S with TDI-8304, of human constitutive proteasome with TDI-8304, and of Pf20Sß6A117D with WLW-vs that give insights into the species selectivity of TDI-8304, resistance to it, and the collateral sensitivity associated with resistance, including that TDI-8304 binds ß2 and ß5 in wild type Pf20S as well as WLW-vs binds ß2 and ß5 in Pf20Sß6A117D. We further show that TDI-8304 kills P. falciparum as quickly as chloroquine and artemisinin and is active against P. cynomolgi at the liver stage. This increases interest in using these structures to facilitate the development of Pf20S inhibitors that target multiple proteasome subunits and limit the emergence of resistance.

Screening and prenatal diagnosis of survival motor neuron gene deletion in pregnant women in Zhaoqing city, Guangdong Province.

OBJECTIVE: A total of 5,200 pregnant women in Zhaoqing city, Guangdong Province, were screened to identify spinal muscular atrophy (SMA) mutation carriers to guide the prevention of SMA and prevent the birth of children with SMA. METHODS: Exons 7 and 8 (E7 and E8) of the survival motor neuron (SMN) 1 gene were detected in women using real-time fluorescence quantitative polymerase chain reaction. SMN1 and SMN2 copy numbers in those who were initially identified as carriers were verified via targeted region capture and next-generation sequencing. When both partners were identified as carriers, prenatal diagnosis of the fetus was performed. RESULTS: Among the screened women, 75 SMA carriers (71 cases had both E7 and E8 heterozygous deletions and 4 cases only had an E7 heterozygous deletion) were identified, with a carrier frequency of 1.44% (95% confidence interval: 1.31-1.65%). Three couples where both spouses were identified as SMA carriers, and their three fetuses were subjected to prenatal genetic analysis. Of the three, one had homozygous deletions of E7 and E8 and the other two had heterozygous deletions of E7 and E8. After a detailed prenatal consultation, the former couple decided to terminate the pregnancy. CONCLUSION: Through screening and prenatal diagnosis of pregnant women in Zhaoqing city, Guangdong Province, the incidence of SMA can be reduced, prevention of birth defects can be improved, incidence of birth defects can be effectively minimized.