Defects in myosin VIIa lead to developmental anomalies of the auditory and visual sensory cells. We sought proteins interacting with the myosin VIIa tail by using the yeast two-hybrid system. Here, we report on shroom2, a submembranous PDZ domain-containing protein that is associated with the tight junctions in multiple embryonic and adult epithelia. Shroom2 directly interacts with the C-terminal MyTH4-FERM domain of myosin VIIa and with F-actin. In addition, a shroom2 fragment containing the region of interaction with F-actin was able to protect actin filaments from cytochalasin-D-induced disruption in MDCK cells. Transfection experiments in MDCK and LE (L fibroblasts that express E-cadherin) cells led us to conclude that shroom2 is targeted to the cell-cell junctions in the presence of tight junctions only. In Ca²⁺-switch experiments on MDCK cells, ZO-1 (also known as TJP1) preceded GFP-tagged shroom2 at the differentiating tight junctions. ZO-1 directly interacts with the serine- and proline-rich region of shroom2 in vitro. Moreover, the two proteins colocalize in vivo at mature tight junctions, and could be coimmunoprecipitated from brain and cochlear extracts. We suggest that shroom2 and ZO-1 form a tight-junction-associated scaffolding complex, possibly linked to myosin VIIa, that bridges the junctional membrane to the underlying cytoskeleton, thereby contributing to the stabilization of these junctions.

Excessive release of inflammatory/pain mediators from peripheral sensory afferents renders nerve endings hyper-responsive, causing central sensitization and chronic pain. Herein, the basal release of proinflammatory calcitonin gene-related peptide (CGRP) was shown to increase the excitability of trigeminal sensory neurons in brainstem slices via CGRP1 receptors because the effect was negated by an antagonist, CGRP8–37. This excitatory action could be prevented by cleaving synaptosomal-associated protein ofM_r25,000 (SNAP-25) with botulinum neurotoxin (BoNT) type A, a potent inhibitor of exocytosis. Strikingly, BoNT/A proved unable to abolish the CGRP1 receptor-mediated effect of capsaicin, a nociceptive TRPV1 stimulant, or its elevation of CGRP release from trigeminal ganglionic neurons (TGNs) in culture. Although the latter was also not susceptible to BoNT/E, apparently attributable to a paucity of its acceptors (glycosylated synaptic vesicle protein 2 A/B), this was overcome by using a recombinant chimera (EA) of BoNT/A and BoNT/E. It bound effectively to the C isoform of SV2 abundantly expressed in TGNs and cleaved SNAP-25, indicating that its /A binding domain (H_C) mediated uptake of the active /E protease. The efficacy of /EA is attributable to removal of 26 C-terminal residues from SNAP-25, precluding formation of SDS-resistant SNARE complexes. In contrast, exocytosis could be evoked after deleting nine of the SNAP-25 residues with /A but only on prolonged elevation of [Ca²⁺]_iwith capsaicin. This successful targeting of /EA to nociceptive neurons and inhibition of CGRP releasein vitroandin situhighlight its potential as a new therapy for sensory dysmodulation and chronic pain.

Transient receptor potential (TRP) A1 and V1 channels relay sensory signals, yet little is known about their transport to the plasmalemma during inflammation. Herein, TRPA1 and TRPV1 were found on vesicles containing calcitonin gene-related peptide (CGRP), accumulated at sites of exo- and endo-cytosis and co-localised on fibres and cell bodies of cultured sensory neurons expressing both. A proinflammatory cytokine, TNFα, elevated their surface content and both resided in close proximity, indicating co-trafficking. Syntaxin 1–interacting protein, Munc18–1, proved necessary for the response to TNFα and for TRPV1-triggered CGRP release. TNFα-induced surface trafficking of TRPV1 and TRPA1 required a synaptic vesicle membrane protein VAMP1 (but not 2/3), which is essential for CGRP exocytosis from large dense-core vesicles. Inactivation of two proteins on the presynaptic plasma membrane, syntaxin-1 or SNAP-25, by botulinum neurotoxin (BoNT)/C1 or /A inhibited the TNFα-elevated delivery. Accordingly, enhancement by TNFα of Ca²⁺ influx through the upregulated surface-expressed TRPV1 and TRPA1 channels was abolished by BoNT/A. Thus, in addition, the neurotoxins’ known inhibition of the release of pain transmitters, their therapeutic potential is augmented by lowering the exocytotic delivery of transducing channels and the resultant hyper-sensitisation in inflammation.

Atopic dermatitis (AD) is a multifaceted, chronic relapsing inflammatory skin disease that affects people of all ages. It is characterized by chronic eczema, constant pruritus, and severe discomfort. AD often progresses from mild annoyance to intractable pruritic inflammatory lesions associated with exacerbated skin sensitivity. The T helper-2 (Th2) response is mainly linked to the acute and subacute phase, whereas Th1 response has been associated in addition with the chronic phase. IL-17, IL-22, TSLP, and IL-31 also play a role in AD. Transient receptor potential (TRP) cation channels play a significant role in neuroinflammation, itch and pain, indicating neuroimmune circuits in AD. However, the Th2-driven cutaneous sensitization of TRP channels is underappreciated. Emerging findings suggest that critical Th2-related cytokines cause potentiation of TRP channels, thereby exaggerating inflammation and itch sensation. Evidence involves the following: (i) IL-13 enhances TRPV1 and TRPA1 transcription levels; (ii) IL-31 sensitizes TRPV1 via transcriptional and channel modulation, and indirectly modulates TRPV3 in keratinocytes; (iii) The Th2-cytokine TSLP increases TRPA1 synthesis in sensory neurons. These changes could be further enhanced by other Th2 cytokines, including IL-4, IL-25, and IL-33, which are inducers for IL-13, IL-31, or TSLP in skin. Taken together, this review highlights that Th2 cytokines potentiate TRP channels through diverse mechanisms under different inflammatory and pruritic conditions, and link this effect to distinct signaling cascades in AD. This review strengthens the notion that interrupting Th2-driven modulation of TRP channels will inhibit transition from acute to chronic AD, thereby aiding the development of effective therapeutics and treatment optimization.

Hyperexcitability disorders of cholinergically innervated muscles are treatable with botulinum neurotoxin (BoNT) A. The seven serotypes (A–G) potently block neurotransmission by binding to presynaptic receptors, undergoing endocytosis, transferring to the cytosol, and inactivating proteins essential for vesicle fusion. Although BoNT/A and BoNT/E cleave SNAP-25, albeit at distinct sites, BoNT/E blocks neurotransmission faster and more potently. To identify the domains responsible for these characteristics, the C-terminal heavy chain portions of BoNT/A and BoNT/E were exchanged to create chimeras AE and EA. After high yield expression in Escherichia coli, these single chain chimeras were purified by two-step chromatography and activated by conversion to disulfide-linked dichains. In vitro, each entered neurons, cleaved SNAP-25, and blocked neuromuscular transmission while causing flaccid paralysis in vivo. Acidification-dependent translocation of the light chain to the cytosol occurred more rapidly for BoNT/E and EA than for BoNT/A and AE because the latter pair remained susceptible for longer to inhibitors of the vesicular proton pump, and BoNT/A proved less sensitive. The receptor-binding and protease domains do not seem to be responsible for the speeds of intoxication; rather the N-terminal halves of their heavy chains are implicated, with dissimilar rates of cytosolic transfer of the light chains being due to differences in pH sensitivity. AE produced the most persistent muscle weakening and therefore has therapeutic potential. Thus, proof of principle is provided for tailoring the pharmacological properties of these toxins by protein engineering.

Blockade of neurotransmitter release by botulinum neurotoxin type A (BoNT_A) underlies the severe neuroparalytic symptoms of human botulism, which can last a few years. The structural basis for this remarkable persistence remains unclear. Herein, recombinant BoNT_A was found to match the neurotoxicity of that from Clostridium botulinum, producing persistent cleavage of synaptosomal-associated protein of 25 kDa (SNAP-25) and neuromuscular paralysis. When two leucines near the C terminus of the protease light chain of A (LC_A) were mutated, its inhibition of exocytosis was followed by fast recovery of intact SNAP-25 in cerebellar neurons and neuromuscular transmission in vivo. Deletion of 6–7 N terminus residues diminished BoNT_A activity but did not alter the longevity of its SNAP-25 cleavage and neuromuscular paralysis. Furthermore, genetically fusing LC_E to a BoNT_A enzymically inactive mutant (BoTIM_A) yielded a novel LC_E-BoTIM_A protein that targets neurons, and the BoTIM_A moiety also delivers and stabilizes the inhibitory LC_E, giving a potent and persistent cleavage of SNAP-25 with associated neuromuscular paralysis. Moreover, its neurotropism was extended to sensory neurons normally insensitive to BoNT_E. LC_E-BoTIM_A(AA) with the above-identified dileucine mutated gave transient neuromuscular paralysis similar to BoNT_E, reaffirming that these residues are critical for the persistent action of LC_E-BoTIM_A as well as BoNT_A. LC_E-BoTIM_A inhibited release of calcitonin gene-related peptide from sensory neurons mediated by transient receptor potential vanilloid type 1 and attenuated capsaicin-evoked nociceptive behavior in rats, following intraplantar injection. Thus, a long acting, versatile composite toxin has been developed with therapeutic potential for pain and conditions caused by overactive cholinergic nerves.