Autism Spectrum Disorders (ASDs) are neurodevelopmental disorders (NDDs) in which children display differences in social interaction/communication and repetitive stereotyped behaviors along with variable associated features. Cul3, a gene linked to ASD, encodes CUL3 (CULLIN-3), a protein that serves as a key component of a ubiquitin ligase complex with unclear function in neurons. Cul3 homozygous deletion in mice is embryonic lethal; thus, we examine the role of Cul3 deletion in early synapse development and neuronal morphology in hippocampal primary neuronal cultures. Homozygous deletion of Cul3 significantly decreased dendritic complexity and dendritic length, as well as axon formation. Synaptic spine density significantly increased, mainly in thin and stubby spines along with decreased average spine volume in Cul3 knockouts. Both heterozygous and homozygous knockout of Cul3 caused significant reductions in the density and colocalization of gephyrin/vGAT puncta, providing evidence of decreased inhibitory synapse number, while excitatory synaptic puncta vGulT1/PSD95 density remained unchanged. Based on previous studies implicating elevated caspase-3 after Cul3 deletion, we demonstrated increased caspase-3 in our neuronal cultures and decreased neuronal cell viability. We then examined the efficacy of the caspase-3 inhibitor Z-DEVD-FMK to rescue the decrease in neuronal cell viability, demonstrating reversal of the cell viability phenotype with caspase-3 inhibition. Studies have also implicated caspase-3 in neuronal morphological changes. We found that caspase-3 inhibition largely reversed the dendrite, axon, and spine morphological changes along with the inhibitory synaptic puncta changes. Overall, these data provide additional evidence that Cul3 regulates the formation or maintenance of cell morphology, GABAergic synaptic puncta, and neuronal viability in developing hippocampal neurons in culture.

Colostrum/Milk is a chief repertoire of antioxidant peptides. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a viable target for Parkinson\'s Disease (PD), as this pathway deduced to be impaired in PD. Cullin-3 is one of the crucial E3 ligase responsible for its regulation. The present study screened peptide libraries of buffalo colostrum & milk peptides for Cullin-3 inhibition, thus ensuing activation of Nrf2 to alleviate the molecular etiopathology in PD using the C. elegans as a model. The structure was modelled, binding sites analyzed and peptide-interactions analyzed by docking. Among the 55 sequences (≤1 kDa), the peptide SFVSEVPEL having the highest dock score (-16.919) was synthesized and evaluated for its effects on oxidative stress markers, antioxidant enzymes, neurochemical marker and Nrf2/Skn-1 levels. The lead peptide alleviated the oxidative pathophysiology and behavioural deficits associated with PD in C. elegans.

We identified Rho-related BTB domain containing 1 (RhoBTB1) as a key regulator of phosphodiesterase 5 (PDE5) activity, and through PDE5, a regulator of vascular tone. We identified the binding interface for PDE5 on RhoBTB1 by truncating full-length RhoBTB1 into its component domains. Co-immunoprecipitation analyses revealed that the C-terminal half of RhoBTB1 containing its two BTB domains and the C-terminal domain (B1B2C) is the minimal region required for PDE5 recruitment and subsequent proteasomal degradation via Cullin-3 (CUL3). The C-terminal domain was essential in recruiting PDE5 as constructs lacking this region could not participate in PDE5 binding or proteasomal degradation. We also identified Pro353 and Ser363 as key amino acid residues in the B1B2C region involved in CUL3 binding to RhoBTB1. Mutation of either of these residues exhibited impaired CUL3 binding and PDE5 degradation, although the binding to PDE5 was preserved. Finally, we employed ascorbate peroxidase 2 (APEX2) proximity labeling using a B1B2C-APEX2 fusion protein as bait to capture unknown RhoBTB1 binding partners. Among several B1B2C-binding proteins identified and validated, we focused on SET domain containing 2 (SETD2). SETD2 and RhoBTB1 directly interacted, and the level of SETD2 increased in response to pharmacological inhibition of the proteasome or Cullin complex, CUL3 deletion, and RhoBTB1-inhibition with siRNA. This suggests that SETD2 is regulated by the RhoBTB1-CUL3 axis. Future studies will determine whether SETD2 plays a role in cardiovascular function.

Tubby-like proteins (TULPs) are characterized by a conserved C-terminal domain that binds phosphoinositides. Collectively, mammalian TULP1-4 proteins play essential roles in intracellular transport, cell differentiation, signaling, and motility. Yet, little is known about how the function of these proteins is regulated in cells. Here, we present the protein-protein interaction network of TULP3, a protein that is responsible for the trafficking of G-protein-coupled receptors to cilia and whose aberrant expression is associated with severe developmental disorders and polycystic kidney disease. We identify several protein interaction nodes linked to TULP3 that include enzymes involved in acetylation and ubiquitination. We show that acetylation of two key lysine residues on TULP3 by p300 increases TULP3 protein abundance and that deacetylation of these sites by HDAC1 decreases protein levels. Furthermore, we show that one of these sites is ubiquitinated in the absence of acetylation and that acetylation inversely correlates with ubiquitination of TULP3. This mechanism is evidently conserved across species and is active in zebrafish during development. Finally, we identify this same regulatory module in TULP1, TULP2, and TULP4 and demonstrate that the stability of these proteins is similarly modulated by an acetylation switch. This study unveils a signaling pathway that links nuclear enzymes to ciliary membrane receptors via TULP3, describes a dynamic mechanism for the regulation of all tubby-like proteins, and explores how to exploit it pharmacologically using drugs.

Speckle-type BTB/POZ protein (SPOP) is a substrate recognition receptor of the cullin-3 (CUL3)/RING type ubiquitin E3 complex. To date, approximately 30 proteins have been identified as ubiquitinated substrates of the CUL3/SPOP complex. Pathologically, missense mutations in the substrate-binding domain of SPOP have been found in prostate and endometrial cancers. Prostate and endometrial cancer-associated SPOP mutations lose and increase substrate-binding ability, respectively. Expression of these SPOP mutants, thus, causes aberrant turnovers of the substrate proteins, leading to tumor formation. Although the molecular properties of SPOP and its cancer-associated mutants have been intensively elucidated, their cellular functions remain unclear. Recently, a number of studies have uncovered the critical role of SPOP and its mutants in DNA damage response and DNA replication. In this review article, we summarize the physiological functions of SPOP as a \"gatekeeper\" of genome stability.

The daily changes of light and dark exemplify a prominent cue for the synchronization of circadian clocks with the environment. The match between external and internal time is crucial for the fitness of organisms, and desynchronization has been linked to numerous physical and mental health problems. Organisms therefore developed complex and not fully understood mechanisms to synchronize their circadian clock to light. In mammals and in Drosophila, both the visual system and non-image-forming photoreceptors contribute to circadian clock resetting. In Drosophila, light-dependent degradation of the clock protein TIMELESS by the blue light photoreceptor Cryptochrome is considered the main mechanism for clock synchronization, although the visual system also contributes. To better understand the visual system contribution, we generated a genetic variant exhibiting extremely slow phototransduction kinetics, yet normal sensitivity. In this variant, the visual system is able to contribute its full share to circadian clock entrainment, both with regard to behavioral and molecular light synchronization. This function depends on an alternative phospholipase C-β enzyme, encoded by PLC21C, presumably playing a dedicated role in clock resetting. We show that this pathway requires the ubiquitin ligase CULLIN-3, possibly mediating CRY-independent degradation of TIMELESS during light:dark cycles. Our results suggest that the PLC21C-mediated contribution to circadian clock entrainment operates on a drastically slower timescale compared with fast, norpA-dependent visual phototransduction. Our findings are therefore consistent with the general idea that the visual system samples light over prolonged periods of time (h) in order to reliably synchronize their internal clocks with the external time.

Protein ubiquitination constitutes a post-translational modification mediated by ubiquitin ligases whereby ubiquitinated substrates are degraded through the proteasomal or lysosomal pathways, or acquire novel molecular functions according to their \"ubiquitin codes.\" Dysfunction of the ubiquitination process in cells causes various diseases such as cancers along with neurodegenerative, auto-immune/inflammatory, and metabolic diseases. KCTD10 functions as a substrate recognition receptor for cullin-3 (CUL3), a scaffold protein in RING-type ubiquitin ligase complexes. Recently, studies by ourselves and others have identified new substrates that are ubiquitinated by the CUL3/KCTD10 ubiquitin ligase complex. Moreover, the type of polyubiquitination (e.g., K27-, K48-, or K63-chain) of various substrates (e.g., RhoB, CEP97, EIF3D, and TRIF) mediated by KCTD10 underlies its divergent roles in endothelial barrier formation, primary cilium formation, plasma membrane dynamics, cell proliferation, and immune response. Here, the physiological functions of KCTD10 are summarized and potential mechanisms are proposed.

Parkinson\'s disease ranks second, after Alzheimer\'s as the major neurodegenerative disorder, for which no cure or disease-modifying therapies exist. Ample evidence indicate that PD manifests as a result of impaired anti-oxidative machinery leading to neuronal death wherein Cullin-3 has ascended as a potential therapeutic target for diseases involving damaged anti-oxidative machinery.
The design of target specific inhibitors for the Cullin-3 protein might be a promising strategy to increase the Nrf2 levels and to decrease the possibility of \"off-target\" toxic properties.
In the present study, an integrated computational and wet lab approach was adopted to identify small molecule inhibitors for Cullin-3. The rational drug designing process comprised homology modeling and derivation of the pharmacophore for Cullin-3, virtual screening of Zinc natural compound database, molecular docking and Molecular dynamics based screening of ligand molecules. In vivo validations of an identified lead compound were conducted in the PD model of C. elegans.
Our strategy yielded a potential inhibitor; (Glide score = -12.31), which was evaluated for its neuroprotective efficacy in the PD model of C. elegans. The inhibitor was able to efficiently defend against neuronal death in PD model of C. elegans and the neuroprotective effects were attributed to its anti-oxidant activities, supported by the increase in superoxide dismutase, catalase and the diminution of acetylcholinesterase and reactive oxygen species levels. In addition, the Cullin-3 inhibitor significantly restored the behavioral deficits in the transgenic C. elegans.
Taken together, these findings highlight the potential utility of Cullin-3 inhibition to block the persistent neuronal death in PD. Further studies focusing on Cullin-3 and its mechanism of action would be interesting.

Wnt signaling pathways direct key physiological decisions in development. Here, we establish a role for a pleckstrin homology domain-containing protein, PLEKHA4, as a modulator of signaling strength in Wnt-receiving cells. PLEKHA4 oligomerizes into clusters at PI(4,5)P2-rich regions of the plasma membrane and recruits the Cullin-3 (CUL3) E3 ubiquitin ligase substrate adaptor Kelch-like protein 12 (KLHL12) to these assemblies. This recruitment decreases CUL3-KLHL12-mediated polyubiquitination of Dishevelled, a central intermediate in canonical and non-canonical Wnt signaling. Knockdown of PLEKHA4 in mammalian cells demonstrates that PLEKHA4 positively regulates canonical and non-canonical Wnt signaling via these effects on the Dishevelled polyubiquitination machinery. In vivo knockout of the Drosophila melanogaster PLEKHA4 homolog, kramer, selectively affects the non-canonical, planar cell polarity (PCP) signaling pathway. We propose that PLEKHA4 tunes the sensitivities of cells toward the stimulation of Wnt or PCP signaling by sequestering a key E3 ligase adaptor controlling Dishevelled polyubiquitination within PI(4,5)P2-rich plasma membrane clusters.

Viral infection of cells is sensed by pathogen recognition receptors that trigger an antiviral innate immune response, and consequently viruses have evolved countermeasures. Vaccinia virus (VACV) evades the host immune response by expressing scores of immunomodulatory proteins. One family of VACV proteins are the BTB-BACK (broad-complex, tram-trac, and bric-a-brac [BTB] and C-terminal Kelch [BACK]) domain-containing, Kelch-like (BBK) family of predicted cullin-3 E3 ligase adaptors: A55, C2, and F3. Previous studies demonstrated that gene A55R encodes a protein that is nonessential for VACV replication yet affects viral virulence in vivo Here, we report that A55 is an NF-κB inhibitor acting downstream of IκBα degradation, preventing gene transcription and cytokine secretion in response to cytokine stimulation. A55 targets the host importin α1 (KPNA2), acting to reduce p65 binding and its nuclear translocation. Interestingly, while A55 was confirmed to coprecipitate with cullin-3 in a BTB-dependent manner, its NF-κB inhibitory activity mapped to the Kelch domain, which alone is sufficient to coprecipitate with KPNA2 and inhibit NF-κB signaling. Intradermal infection of mice with a virus lacking A55R (vΔA55) increased VACV-specific CD8+ T-cell proliferation, activation, and cytotoxicity in comparison to levels of the wild-type (WT) virus. Furthermore, immunization with vΔA55 induced increased protection to intranasal VACV challenge compared to the level with control viruses. In summary, this report describes the first target of a poxvirus-encoded BBK protein and a novel mechanism for DNA virus immune evasion, resulting in increased CD8+ T-cell memory and a more immunogenic vaccine.IMPORTANCE NF-κB is a critical transcription factor in the innate immune response to infection and in shaping adaptive immunity. The identification of host and virus proteins that modulate the induction of immunological memory is important for improving virus-based vaccine design and efficacy. In viruses, the expression of BTB-BACK Kelch-like (BBK) proteins is restricted to poxviruses and conserved within them, indicating the importance of these proteins for these medically important viruses. Using vaccinia virus (VACV), the smallpox vaccine, we report that the VACV BBK protein A55 dysregulates NF-κB signaling by disrupting the p65-importin interaction, thus preventing NF-κB translocation and blocking NF-κB-dependent gene transcription. Infection with VACV lacking A55 induces increased VACV-specific CD8+ T-cell memory and better protection against VACV challenge. Studying viral immunomodulators therefore expands not only our understanding of viral pathogenesis and immune evasion strategies but also of the immune signaling cascades controlling antiviral immunity and the development of immune memory.