Structure of mammalian V-ATPase with the TLDc domain protein mEAK7 bound

V-ATPases are rotary proton pumps that serve as signaling hubs, with numerous proposed binding partners in cells. We used cryoEM to detect endogenous proteins that associate with V-ATPase from porcine kidney. A super-stoichiometric copy of subunit C was found in ~3% of complexes, while an additional ~1.6% of complexes bound mEAK7, a protein with proposed roles in dauer formation in nematodes and mTOR signaling in mammals. High-resolution cryoEM of porcine kidney V-ATPase with recombinant mEAK7 shows that mEAK7's TLDc domain, which is found in other proteins proposed to bind V-ATPase, interacts with V-ATPase's stator while its C-terminal α helix binds V-ATPase's rotor. This crosslink would be expected to inhibit rotary catalysis. However, exogenous mEAK7 does not inhibit purified V-ATPase activity and mEAK7 overexpression in cells does not alter lysosomal or phagosomal pH. Instead, cryoEM suggests that interaction of mEAK7 with V-ATPase is disrupted by ATP-induced rotation of the rotor. Together, these results reveal how TLDc domains bind V-ATPases and suggest that V-ATPase binding proteins can form labile interactions that are sensitive to the enzyme's activity.

Low-concentration T-2 Toxin Attenuates Pseudorabies Virus Replication in Porcine Kidney 15 Cells

Pseudorabies, caused by pseudorabies virus (PRV), is the main highly infectious disease that severely affects the pig industry globally. T-2 toxin (T2), a significant mycotoxin, is widely spread in food and feeds and shows high toxicity to mammals. The potential mechanism of the interaction between viruses and toxins is of great research value because revealing this mechanism may provide new ideas for their joint prevention and control. In this study, we investigated the effect of T2 on PRV replication and the mechanism of action. The results showed that at a low dose (10 nM) T2 had no significant effect on porcine kidney 15 (PK15) cell viability. However, this T2 concentration alleviated PRV-induced cell injury and increased cell survival time. Additionally, the number of PK15 cells infected with PRV was significantly reduced by T2 treatment. Similarly, T2 significantly decreased the copy number of PRV. Investigation of the mechanism revealed that 10 nM T2 significantly inhibited PRV replication and led to downregulation of oxidative stress- and apoptosis-related genes. These results suggest that oxidative stress and apoptosis are involved in the inhibition of PRV replication in PK15 cells by low-concentration T2. Taken together, we demonstrated the protective effects of T2 against PRV infection. A low T2 concentration inhibited the replication of PRV in PK15 cells, and this process was accompanied by downregulation of by the oxidative stress and apoptosis signaling pathways. Our findings partly explain the interaction mechanism between T2 and PRV, relating to oxidative stress and apoptosis, though further research is required.

Integrative Omics Reveals Subtle Molecular Perturbations Following Ischemic Conditioning in a Porcine Kidney Transplant Model

ABSTRACTBackgroundRemote Ischemic Conditioning (RIC) has been proposed as a therapeutic intervention to circumvent the Ischemia/reperfusion injury (IRI) that is inherent to organ transplantation. Using a porcine kidney transplant model, we aimed to decipher the subclinical molecular effects of a RIC regime, compared to non-RIC controls.MethodsKidney pairs (n = 8+8) were extracted from brain dead donor pigs and transplanted in juvenile recipient pigs following a period of cold ischemia. One of the two kidney recipients in each pair was subjected to RIC prior to kidney graft reperfusion, while the other served as non-RIC control. We designed an advanced integrative Omics strategy combining transcriptomics, proteomics, and phosphoproteomics to deduce molecular signatures in kidney tissue that could be attributed to RIC.ResultsIn kidney grafts taken out 10 h after transplantation we detected minimal molecular perturbations following RIC compared to non-RIC at the transcriptome level, but more pronounced effects at the proteome level. In particular, we noted that RIC resulted in suppression of tissue inflammatory profiles. Furthermore, an accumulation of muscle extracellular matrix assembly proteins in kidney tissues was detected at the protein level, which may be in response to muscle tissue damage and/or fibrosis.ConclusionsOur data identifies subtle molecular phenotypes in porcine kidneys following RIC and this knowledge could potentially aid optimisation of remote ischaemia protocols in renal transplantation.