"Decreased SUMOylation of USP5 after nerve injury would aid this process such that USP5 interactions with Cav3.2 are enhanced when USP5 SUMOylation is decreased."
"Here, we describe the development of an ELISA-based assay to screen a library of pharmacologically active compounds (including clinically used drugs) for molecules capable of disrupting the USP5-Cav3.2 interaction."
"No significant effects on current amplitude or biophysical properties of the channel were observed (7±2% inhibition (n=3) and 3±1% inhibition (n=3) of channel activity for suramin and gossypetin, respectively, which is indistinguishable from rundown), consistent with an action on the USP5-Cav3.2 channel interaction rather than direct effects on Cav3.2 channel function."
"In contrast, co-immunoprecipitation experiments revealed that intrathecal administration of interleukin-1 beta in wild-type mice led to an increase in the interaction between USP5 and Cav3.2 in the spinal dorsal horn."
"Uncoupling the Cav3.2-USP5 interaction produces analgesia in vivo , and these advances pave the way for other conceptually similar approaches that focus on a divergent molecular target [ xref – xref ]."
"The biflavenoid gossypetin (318 Da) and the polysulphonated naphtylurea suramin (~1.3 kDa) are vastly different in size, and yet both compounds effectively disrupted USP5 binding to the Cav3.2 III-IV linker region."
"The acute sensitization initiated by optical stimulation (10 Hz) of TRPV1-ChR2 neurons, is sufficient to increase USP5 expression, which results in increased Cav3.2 T-type activity and increased mechanical hypersensitivity that is dependent on the interaction of USP5 and Cav3.2 [ xref ]."
"While our Tat peptide approach delivered proof of concept for targeting the Cav3.2-USP5 interaction as a strategy for targeting various pain conditions, small organic mimetics of these peptides are a preferred strategy for therapeutics."
"We therefore developed an ELISA-based assay for identifying small organic disruptors of the Cav3.2-USP5 interaction."
"To determine which part of USP5 interacts with Cav3.2 channels, we designed short peptides (35–45 mers) corresponding to each of the major USP5 domains xref , with each peptide containing at least one α-helix."
"Given the relevance of the pro-inflammatory cytokine interleukin-1 beta in many forms of pathological pain, we hypothesized that interleukin-1 beta may be a critical cofactor required to drive upregulation of interactions between USP5 and Cav3.2 channels."
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"Altogether, these data reveal suramin and gossypetin as potential small organic disruptors of USP5 interactions with Cav3.2."
"While the above findings support the hypothesis that IL-1β drives upregulation of T-type channels via USP5 to induce pain, these effects were studied under acute conditions (<1 h) that yield transient pain (∼15 min) and may be secondary to interactions between IL-1β and glial cells, which are known to express IL-1RI. xref , xref To study neuroinflammatory interaction more directly, while reflecting sustained release after physical injury, we used DRG neuron cultures to evaluate the level of USP5 bound to Cav3.2 in response to overnight (∼24 h) exposure to IL-1β."
"Suramin inhibited USP5 binding to Cav3.2 channels obtained from ob/ob mouse dorsal horns while total Cav3.2 protein levels remained similar (Figure xref B, C)."
"Here we describe the regulation of the Cav3.2-USP5 interaction by SUMOylation."
"t. injection of IL-1β (0.1 pg), co-immunoprecipation (co-IP) experiments demonstrated that IL-1β increases the interaction between USP5 and Cav3.2 in the spinal dorsal horn, relative to vehicle control (PBS) mice ( xref and ( xref ))."
"We then sought to determine whether SUMOylation of USP5 affects the interaction between USP5 and Cav3.2 channels."
"Next, we determined if suramin altered endogenous USP5-Cav3.2 protein interactions in the dorsal horns from diabetic and non-diabetic mice."
"The latter is reminiscent of the hydroxylated chromene core of gossypetin, thus suggesting the possibility that the disruption of USP5-Cav3.2 interactions may involve this bicyclic aromatic structure."
"Consistent with our observations, this region has been previously described as a potential site for substrate targeting and specificity. xref In contrast, this region does not appear to be important for substrate modification by the catalytic site because deletion of the cUBP (163–291) domain did not affect the hydrolysis rate of ubiquitin-AMC. xref Together with our previous identification of a short (∼20 amino acid) stretch of residues in the domain III-IV linker of the Cav3.2 channel, xref we now have two complementary tools that allow us to disrupt USP5-Cav3.2 interactions both in vitro and in vivo."
"In support of such a mechanism, we discovered that, by sustaining elevated levels of IL-1β in DRG cultures, interactions between USP5 and Cav3.2 can be maintained."
"Altogether, our findings identify interleukin-1 beta as an upstream trigger for the upregulation of interactions between USP5 and Cav3.2 channels in the pain pathway, presumably by triggering increased firing activity in afferent fibers."
"Overall, our results indicate that disrupting the interaction between the deubiquitinase USP5 and Cav3.2 calcium channels via a small organic molecule is a promising new strategy for treating a spectrum of chronic pain states."
"This experiment suggests that SUMOylation state of USP5 can regulate USP5 interactions with Cav3.2 calcium channels."
"Suramin and gossypetin inhibited USP5 binding to Cav3.2 channels by 50-60% at 5 μM (Figure xref A-D)."
"Overall, our observations extend our previous findings showing that peripheral nerve injury upregulates USP5 levels and leads to an enhanced interaction between USP5 and Cav3.2 [ xref ]."
No evidence text available
"It is interesting that in a preliminary in vitro screen of potential mediators, which included brain-derived neurotrophic factor, nerve growth factor, and tumor necrosis factor-alpha, none were as effective as IL-1β at inducing interactions between USP5 and Cav3.2 (data not shown)."
"Hence, preventing deSUMOylation of USP5 could provide a strategy for enhancing USP5 interactions with Cav3.2, which would in turn be predicted to lead to analgesia."
"Since this process is relevant to both inflammatory and neuropathic pain states, xref , xref we initially proposed that neural activity may be a common trigger. xref This idea was supported by observations that activity stemming from optogenetically targeted cutaneous nociceptors alone is sufficient to upregulate USP5 expression, and therefore to increase interactions between USP5 and Cav3.2. xref However, this activity-dependent phenomenon is not self-sustaining, instead promoting only a transient pain state that cannot account for the upregulation observed after injury."
"We have previously reported that pain hypersensitivity in diabetic ob/ob mice can be reversed by blocking the interactions between USP5 and Cav3.2 by small organic molecule mimetics. xref To determine whether the TAT-cUBP1-USP5 peptide was similarly effective, we assessed thermal withdrawal threshold in ob/ob mice before and after delivery of the TAT-cUBP1-USP5 peptide."
"Here, we report the effects of SUMOylation on USP5 interactions with Cav3.2 calcium channels."
"Screening for modulators of the USP5-Cav3.2 interaction."
"Under these conditions and relative to vehicle control (0.1% BSA in PBS), IL-1β produced a clear increase in the level of interaction between USP5 and Cav3.2 channels in co-IP experiments ( xref and ( xref )), which is mediated by IL-1RI, as this effect was almost completely attenuated in the presence of IL-1Ra (100 ng/ml)."
"In summary, we have extended our previous work on USP5-Cav3.2 channel interactions to identify a key structural USP5 domain that is responsible for the actions of USP5 on this channel isoform."