Molecular basis of USP7 inhibition by selective small-molecule inhibitors.

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers.

An improved cell-based method for determining the γ-secretase enzyme activity against both Notch and APP substrates.

γ-Secretase modulators (GSM), which reduce amyloidogenic Aβ(42) production while maintaining total Aβ levels, and Notch-sparing γ-secretase inhibitors (GSIs) are promising therapies for the treatment of Alzheimer's Disease (AD). To have a safety margin for therapeutic use, GSMs and GSIs need to allow Notch intracellular domain (NICD) production, while preventing neurotoxic Aβ peptide production. Typically, GSI and GSM effects on these substrates are determined using two different cell lines, one for the measurement of enzyme activity against each substrate.

Heteropoda toxin 2 interaction with Kv4.3 and Kv4.1 reveals differences in gating modification.

Kv4 (Shal) potassium channels are responsible for the transient outward K(+) currents in mammalian hearts and central nervous systems. Heteropoda toxin 2 (HpTx2) is an inhibitor cysteine knot peptide toxin specific for Kv4 channels that inhibits gating of Kv4.3 in the voltage-dependent manner typical for this type of toxin.

How strict is the correlation between STIM1 and Orai1 expression, puncta formation, and ICRAC activation?

Stromal interaction molecule 1 (STIM1) and Orai1 have been identified as crucial elements of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of their functional interaction remains controversial. It is now well established that, upon depletion of the stores, both molecules can accumulate and colocalize in specific areas (puncta) where the endoplasmic reticulum comes in close proximity to the plasma membrane. Some models propose a direct interaction between STIM1 and Orai1 as the most straightforward mechanism for signal transduction from the stores to the plasma membrane.

Store-operated Orai1 and IP3 receptor-operated TRPC1 channel.

Store-operated channels (SOC) are known to be physiologically activated following agonist-induced IP3 production and depletion of Ca2+ stores. Here we present molecular,biophysical and mechanistic evidence that two ubiquitously expressed plasma membrane channels may be responsible for creating a complex and sometimes controversial SOC image: one being a real SOC encoded by Orai1 and activated exclusively upon depletion of Ca2+ stores (via iPLA2beta -dependent pathway), while the second one is an IP3 receptor-operated channel (IP3ROC) encoded by TRPC1 and activated via its conformational coupling with IP3 receptor. In RBL-2H3 cells endogenously expressing Orai1 and TRPC1, we unmasked and characterized whole-cell current through IP3ROC channels that was hiding behind some familiar fingerprints of ICRAC, a current through the classical Ca2+-selective SOC (CRAC) channels.

Novel role for STIM1 as a trigger for calcium influx factor production.

STIM1 has been recently identified as a Ca(2+) sensor in endoplasmic reticulum (ER) and an initiator of the store-operated Ca(2+) entry (SOCE) pathway, but the mechanism of SOCE activation remains controversial. Here we focus on the early ER-delimited steps of the SOCE pathway and demonstrate that STIM1 is critically involved in initiating of production of calcium influx factor (CIF), a diffusible messenger that can deliver the signal from the stores to plasma membrane and activate SOCE. We discovered that CIF production is tightly coupled with STIM1 expression and requires functional integrity of its intraluminal sterile alpha-motif (SAM) domain.

Heteropoda toxin 2 is a gating modifier toxin specific for voltage-gated K+ channels of the Kv4 family.

Kv4 voltage-gated K(+) channels are responsible for transient K(+) currents in the central nervous system and in the heart. HpTx2 is a peptide toxin that selectively inhibits these currents; making it a useful probe for understanding Kv4 channel structure and drug binding. Therefore, we developed a method to produce large amounts of recombinant HpTx2.