Recombinant Pongo abelii Serine/threonine-protein kinase ULK4 (ULK4), partial

What is the molecular structure of ULK4 and how does it differ from other ULK family members?

ULK4 is a large neuron-specific 142 kDa polypeptide consisting of an N-terminal pseudokinase domain and 5 C-terminal HEAT repeats that function in scaffolding and protein-protein interactions . Unlike other ULK family members (ULK1, ULK2, and ULK3) which possess catalytic activity, ULK4 is a pseudokinase with unusual mutations in the kinase catalytic motifs .

The most striking structural feature of ULK4 is that instead of the lysine:glutamate salt bridge (one of the most conserved features in protein kinases), ULK4 contains a tryptophan:leucine hydrophobic interaction . This appears to be a unique feature as this is possibly the only hydrophobic bridge in the entire kinome. The hydrophobic residue at position 33 of ULK4 is evolutionarily conserved across species, while the Trp 46 is mostly conserved except in fish, where it is a histidine residue .

How does ULK4 bind ATP despite lacking canonical kinase motifs?

Although ULK4 lacks the typical sequence motifs required for ATP binding in kinases, high-resolution structural studies have revealed that it can still tightly bind ATP through an unusual mechanism . ULK4 demonstrates a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC .

Binding studies using Mant-ADP (a fluorescent analog of ADP) have produced binding constants of ULK4 with different nucleotides:

Interestingly, the presence of the co-factor ATP is required for structural stability of ULK4 . This suggests that despite lacking catalytic activity, ATP binding plays an important structural role in ULK4 function.

What signaling pathways does ULK4 influence and how?

Despite being catalytically inactive, ULK4 influences several major signaling pathways critical for neurodevelopment. Knockdown of ULK4 alters the activity of:

• Wnt signaling pathway

• Protein kinase C (PKC) pathway

• Mitogen-activated protein kinase (MAPK) pathway

• Extracellular signal-regulated kinase (ERK1/2) pathway

• c-Jun N-terminal kinases (JNK) pathway

ULK4 can form a functional interactome by physically binding with PP2A and PP1α, the two most abundant phosphatases responsible for over 90% of total Ser/Thr dephosphorylation in eukaryotes . Through this interaction, ULK4 regulates the expression of p-Akt and p-GSK-3α/β, which are critical signaling molecules in neuronal development and function .

How does ULK4 regulate neurogenesis and corticogenesis?

ULK4 plays a crucial role in neurogenesis during developmental stages, with both in vivo and in vitro studies demonstrating its importance in neural cell proliferation and cortical development:

• ULK4 is widely expressed in the ventricular zone (VZ), subventricular zone (SVZ), and cortical plate in the developing mouse brain (E15.5), and in all cortical layers after postnatal day 7

• Knockdown of ULK4 at E15.5 significantly inhibits cell proliferation and corticogenesis in mice

• ULK4 null knockout mice show a reduced neural stem cell pool in the forebrain at birth, which is important for adult neurogenesis

• ULK4 expression is cell cycle-dependent, with peak expression in the G2/M phases

The mechanism appears to involve regulation of the Wnt signaling pathway, as ULK4 mutant mice show decreased neurogenesis during both embryonic and adult stages, likely due to dysregulated Wnt signaling .

What experimental approaches effectively demonstrate ULK4's role in neurite development?

Several complementary experimental approaches have revealed ULK4's critical function in neurite branching and neuronal migration:

• Cell Culture Models: Knockdown of ULK4 in SH-SY5Y neuroblastoma cells leads to reduced expression of acetylated α-tubulin, resulting in decreased dendrite length and branching, and compromised neuronal migration

• In Utero Electroporation: This approach demonstrated that ULK4 knockdown causes perturbed neurite arborization in the pyramidal neurons of the cortex

• ULK4 Knockout Mice: Analysis of these models reveals compromised neurite development and function

These methodologies collectively demonstrate that ULK4 regulates neurite development through multiple signaling pathways including PKC, MAPK, ERK, and JNK pathways .

How does ULK4 contribute to myelination and white matter integrity?

ULK4 has been identified as a crucial factor for myelination and white matter integrity through studies of ULK4−/− mice, which exhibit:

• 50% decrease in myelination

• General reduction in myelin components

• Thin axons

• Extensive neuroinflammation that promotes hypomyelination

At the molecular level, ULK4 deficiency significantly attenuates:

• Enrichment of oligodendrocyte transcription factors

• Formation of new oligodendrocytes

• Development of myelinating oligodendrocytes

These findings explain why ULK4 mutant mice demonstrate compromised cognitive performance, as myelin produced by oligodendrocytes controls impulse conduction speed along axons, which is critical for cognitive function.

What genetic evidence links ULK4 to schizophrenia and other neuropsychiatric disorders?

Multiple genetic studies have implicated ULK4 in several neuropsychiatric disorders:

• Four schizophrenia patients with ULK4 intragenic deletions (spanning exons 21-34) were identified among 3,391 patients

• SNPs rs7651623 and rs2030431 are associated with discontinuation of antipsychotics in schizophrenia patients

• ULK4 deletion is enriched in patients with schizophrenia (2/708), bipolar disorder (2/1,136), and autism (1/507) in the Decode database

• SNPs rs1052501, rs1716975, and rs2272007 (located in exons 2, 7, and 17) show allelic transmission disequilibrium from parents to children with autism spectrum disorder

• SNP rs17210774 is significantly associated with bipolar disorder in Caucasians

• SNP rs1722850 (downstream of ULK4) is related to major depressive disorders

A recent clinical study also revealed cases with ULK4 intragenic microdeletion showing autistic features . The brain-body genetic resource exchange (BBGRE) cohort reported an incidence of 1.2‰ showing ULK4 copy number variation and exhibiting pleiotropic neurodevelopmental problems including learning difficulties and language delay .

What mechanisms link ULK4 dysfunction to schizophrenia pathophysiology?

Several mechanisms appear to connect ULK4 dysfunction to schizophrenia:

• Altered Signaling Pathways: ULK4 knockdown alters Wnt, PKC, MAPK, ERK1/2, and JNK signaling pathways that are commonly dysregulated in schizophrenia

• Neurogenesis Defects: ULK4 knockout and hypomorphic mice present congenital hydrocephalus with dilated ventricles and CSF accumulation. Interestingly, a proportion of schizophrenia patients also display increased global or regional CSF

• Neurotransmitter Imbalance: ULK4 heterozygous mice display anxiety-like behavior with reduced GABAergic neurons in the basolateral amygdala and hippocampus

• White Matter Abnormalities: ULK4−/− mice show significant hypomyelination, which aligns with white matter abnormalities observed in schizophrenia patients

• Reward System Functioning: Functional genetic variation at the ULK4 locus affects the human extended dopaminergic reward system, as demonstrated using functional magnetic resonance imaging (fMRI) during performance of the Desire-Reason-Dilemma (DRD) paradigm

How do ULK4 genetic variants impact brain function in humans?

Recent research has investigated how ULK4 genetic variants affect human brain function:

A study involving 234 participants with functional neuroimaging (fMRI) data examined the effects of genetic variation in the ULK4 gene on reward system functioning using the Desire-Reason-Dilemma (DRD) paradigm . This paradigm allows assessment of brain activation in response to conditioned reward stimuli.

The study demonstrated that functional genetic variation at the ULK4 locus affects the human extended dopaminergic reward system . This finding provides a potential neurobiological mechanism by which ULK4 variants could influence susceptibility to psychiatric disorders characterized by reward processing abnormalities, such as schizophrenia and addiction disorders.

What are effective methods for studying ULK4 binding partners?

Several approaches have proven effective for identifying and validating ULK4 interaction partners:

• BioID Proximity Labeling: This technique has successfully identified high-confidence interactors of the ULK4 pseudokinase and armadillo repeat domains. Many identified interaction partners were centrosomal and tubulin-associated proteins as well as several active kinases, suggesting important regulatory roles for ULK4

• Biochemical Validation: After identification through BioID, putative interactions can be validated using co-immunoprecipitation and pulldown assays

• Fluorescence Resonance Energy Transfer (FRET): ULK4 exhibits strong FRET between the protein and Mant-ADP nucleotide, allowing monitoring of nucleotide binding to determine kinetic parameters and enabling high-throughput screening of inhibitors that can displace Mant-ADP

Studies using these methods have identified key interactions including:

• ULK4 forms an interactome with PP2A and PP1α phosphatases

• ULK4 interacts with Fused/STK36 in a pathway required for stable assembly of motile cilia

• The HEAT repeats of ULK4 interact with CAMSAP1, OFD1, and PAN2

What animal models are available for studying ULK4-related disorders?

Several animal models have been developed to study ULK4 function and dysfunction:

• ULK4 Knockout Mice: Complete knockout of ULK4 in mice leads to:

  • Congenital hydrocephalus

  • Reduced neural stem cell pool

  • Reduced myelination (50% decrease)

• ULK4 Hypomorphic Mice: These mice carry hypomorphic alleles with DNA sequence deletion after exon 7 of ULK4:

  • Show massive disruption of genes related to ciliogenesis

  • Display hydrocephalus

• Conditional ULK4 Knockout Models: Created using the Cre-loxP system, such as Nestin-Cre:Ulk4 fl/fl (Ulk4 fl/fl CKO) mice:

  • Allow tissue-specific deletion of ULK4 in neural stem cells

  • Enable study of ULK4's role in specific neural populations

• Leishmania mexicana ULK4 KO Model: This non-mammalian model has helped elucidate ULK4's role in ciliary/flagellar function:

  • ULK4 KO mutants exhibit structural defects of the flagellum cytoskeleton

  • Demonstrated interaction between ULK4 and Fused/STK36

What screening methods can identify ULK4 inhibitors for research applications?

Despite ULK4 being a pseudokinase, researchers have developed effective screening methods to identify molecules that interact with ULK4:

• Virtual Screening: Computational approaches using high-resolution crystal structures of ULK4:

  • Researchers have screened approximately 3.3 million purchasable lead-like compounds from ZINC15 using both OpenEye and Schrödinger suites

  • Visual inspection focuses on compounds that can make similar contacts to key pharmacophores

• Fluorescence-Based Assays:

  • The Mant-ADP assay monitors FRET between ULK4 protein and the Mant-ADP nucleotide

  • This allows for high-throughput screening of compounds that can displace the fluorescent nucleotide

• Compound Validation:

  • Initial hits are tested at single doses

  • Those showing inhibition undergo dose-response curve analysis

  • Several compounds have demonstrated inhibition of Mant-ADP binding in the double-digit micromolar range

These approaches have identified starting points for ULK4-targeted compounds with some conserved pharmacophores such as the purine and spiro-lactam groups .

How might ULK4-based therapeutic strategies be developed?

Despite ULK4 being a pseudokinase with no catalytic activity, several therapeutic strategies could be developed:

• Small Molecule Modulators: Compounds that bind to ULK4 could modulate its protein-protein interactions or structural stability. Crystal structures of ULK4 provide a foundation for structure-based drug design

• Gene Therapy Approaches: Since ULK4 deletions are linked to neuropsychiatric disorders, gene supplementation strategies could restore proper ULK4 expression. Studies have shown that defects from ULK4 knockdown can be reversed successfully by ULK4 gene supplementation

• Targeting Downstream Pathways: Therapeutic interventions could focus on correcting dysregulated signaling pathways resulting from ULK4 dysfunction, including Wnt, PKC, MAPK, ERK1/2, and JNK pathways

• Interacting Partner Modulation: As ULK4 interacts with various proteins including PP2A and PP1α phosphatases, modulating these interactions could provide alternative therapeutic approaches

The development of ULK4-based therapeutics would benefit from further understanding of its interaction network and the consequences of its dysfunction in relevant cell types and tissues.

What are the critical knowledge gaps in ULK4 research?

Despite significant progress in understanding ULK4's structure and function, several critical knowledge gaps remain:

• Complete Interactome: While some ULK4 binding partners have been identified, a comprehensive map of its interactome across different cell types and developmental stages is lacking

• Tissue-Specific Functions: More research is needed to fully understand ULK4's role in different neural cell types and brain regions

• Mechanistic Connection to Disease: The precise mechanisms by which ULK4 variants contribute to neuropsychiatric disorders remain to be fully elucidated

• Evolutionary Significance: Although evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop that stabilizes the C helix, the evolutionary advantage of this change remains unclear

• Regulatory Mechanisms: How ULK4 expression and function are regulated during development and in response to cellular stressors requires further investigation