Structural and functional validation of a highly specific Smurf2 inhibitor.

Smurf1 and Smurf2 are two closely related member of the HECT (homologous to E6AP carboxy terminus) E3 ubiquitin ligase family and play important roles in the regulation of various cellular processes. Both were initially identified to regulate transforming growth factor-β and bone morphogenetic protein signaling pathways through regulating Smad protein stability and are now implicated in various pathological processes. Generally, E3 ligases, of which over 800 exist in humans, are ideal targets for inhibition as they determine substrate specificity; however, there are few inhibitors with the ability to precisely target a particular E3 ligase of interest.

Apigenin directly interacts with and inhibits topoisomerase 1 to upregulate CD26/DPP4 on colorectal carcinoma cells.

CD26/dipeptidyl peptidase IV (DPP4) is a cell-surface glycoprotein present on most epithelial cells that modulates the local response to external signals. We have previously shown that the dietary flavone apigenin (4',5,7-trihydroxyflavone) upregulates cell-surface CD26/DPP4 on human colorectal carcinoma (CRC) cells and regulates its activities. We observed a unique synergistic interaction with the CRC chemotherapeutic agent irinotecan, which through its metabolite SN38 elevates CD26 at doses that are sub-cytotoxic.

Targeted Degradation of 53BP1 Using Ubiquitin Variant Induced Proximity.

In recent years, researchers have leveraged the ubiquitin-proteasome system (UPS) to induce selective degradation of proteins by E3 ubiquitin ligases, which has great potential as novel therapeutics for human diseases, including cancer and neurodegenerative disorders. However, despite extensive efforts, only a handful of ~600 human E3 ligases were utilized, and numerous protein-protein interaction surfaces on E3 ligases were not explored. To tackle these problems, we leveraged a structure-based protein engineering technology to develop a multi-domain fusion protein bringing functional E3 ligases to the proximity of a target protein to trigger its proteasomal degradation, which we termed Ubiquitin Variant Induced Proximity (UbVIP).

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1.

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery.

Unique cysteine-enriched, D2L5 and D4L6 extracellular loops in Ca3 T-type channels alter the passage and block of monovalent and divalent ions.

Invertebrate LCa3 shares the quintessential features of vertebrate Ca3 T-type channels, with a low threshold of channel activation, rapid activation and inactivation kinetics and slow deactivation kinetics compared to other known Ca channels, the Ca1 and Ca2 channels. Unlike the vertebrates though, Ca3 T-type channels in non-cnidarian invertebrates possess an alternative exon 12 spanning the D2L5 extracellular loop, which alters the invertebrate LCa3 channel into a higher Na and lower Ca current passing channel, more resembling a classical Na1 Na channel. Cnidarian Ca3 T-type channels can possess genes with alternative cysteine-rich, D4L6 extracellular loops in a manner reminiscent of the alternative cysteine-rich, D2L5 extracellular loops of non-cnidarian invertebrates.

Eukaryotic Voltage-Gated Sodium Channels: On Their Origins, Asymmetries, Losses, Diversification and Adaptations.

The appearance of voltage-gated, sodium-selective channels with rapid gating kinetics was a limiting factor in the evolution of nervous systems. Two rounds of domain duplications generated a common 24 transmembrane segment (4 × 6 TM) template that is shared amongst voltage-gated sodium (Na1 and Na2) and calcium channels (Ca1, Ca2, and Ca3) and leak channel (NALCN) plus homologs from yeast, different single-cell protists (heterokont and unikont) and algae (green and brown). A shared architecture in 4 × 6 TM channels include an asymmetrical arrangement of extended extracellular L5/L6 turrets containing a 4-0-2-2 pattern of cysteines, glycosylated residues, a universally short III-IV cytoplasmic linker and often a recognizable, C-terminal PDZ binding motif.

The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+) concentrations, promoting calcium-dependent inactivation of L-type calcium channels.

Kinetic mechanism of the exchanges catalysed by the adenine-nucleotide carrier.

Initial rates of the exchange ADPin/ADPout catalysed by the adenine-nucleotide carrier of rat-heart mitochondria have been studied under conditions where internal and external ADP may be varied. The initial rate was measured within 1 s by the carboxyatractyloside-stop method, using a rapid-mixing technique. The double-reciprocal plots v0(-1) versus [ADP]out-1 at different internal-ADP concentrations and v0(-1) versus [ADP]in-1 at different external-ADP concentrations exhibit straight-line relationships having a common point of intersection on the axis of ordinates.