NUBP2 Human

What is NUBP2 and what protein family does it belong to?

NUBP2 (Nucleotide-binding protein 2), also known as cytosolic Fe-S cluster assembly factor NUBP2, is a protein encoded by the NUBP2 gene in humans. It belongs to the NUBP/MRP gene subfamily of ATP-binding proteins, which are characterized by their nucleotide-binding properties. NUBP2 has structural homology to the cell division inhibitor MinD and is related to the ParA family of proteins. In eukaryotes, there are two main types of these proteins: NUBP1 and NUBP2, which often function in partnership for various cellular processes .

What are the primary cellular functions of NUBP2?

NUBP2 serves multiple critical cellular functions:

• Iron-sulfur cluster biogenesis: NUBP2 works with NUBP1 to facilitate the cytoplasmic iron-sulfur biogenesis pathway. Together, they act upon iron and sulfur atoms to create Fe4-S4 iron-sulfur clusters that serve as catalytic subunits in enzymes like aconitase, which converts citrate to iso-citrate .

• Centrosome regulation: NUBP2 functions as a negative regulator of centrosomes and ciliogenesis. It localizes to the basal body of the primary cilium, which serves as a nucleating center for the extension of the microtubule-based axoneme supporting the primary cilium .

• Protein interactions: NUBP2 interacts with multiple proteins including ACO1 (Iron-responsive element-binding protein 1), MAPK8IP3 (C-jun-amino-terminal kinase-interacting protein 3), IGFALS (Insulin-like growth factor-binding protein complex acid labile chain precursor), KIF11 (Kinesin-like protein KIF11), SEPP1 (Selenoprotein P precursor), and CA1 (Carbonic anhydrase 1) .

How is NUBP2 expressed during mammalian development?

NUBP2 shows distinct expression patterns during development. In mouse models, RNAScope RNA in situ hybridization has demonstrated that Nubp2 is highly expressed throughout the neural epithelium at embryonic day 10.5 (E10.5). This pattern continues at E12.5 and E14.5, with expression becoming more robust in the apical surface where neurogenesis occurs. By E18.5, expression is almost entirely restricted to the ventricular zone of the developing brain. Interestingly, at postnatal day 21 (P21), expression is very low in the cortex but quite prominent in the cerebellum, with robust signaling in the Purkinje cells and inner molecular layer .

This expression pattern suggests NUBP2 has a preferential role in germinal zones of the developing forebrain, consistent with its functions in neurogenesis and stem cell maintenance .

What mouse models are available for studying NUBP2 function?

Researchers have developed several mouse models to study NUBP2 function:

• Conditional knockout models: The Emx1-Cre/wt; Nubp2flox/flox (Nubp2 cKO) model has been used to study forebrain-specific deletion of Nubp2. This model demonstrates significantly smaller forebrains at E18.5 compared to controls, with the difference becoming more pronounced by P23 when mutant brains are approximately 2.5 times smaller than controls. The cortices of these mutants also become distinctly thinner by P1 .

• Compound mutant models: Researchers have also examined the effects of Nubp2 deletion in combination with other genetic modifications. For example, studies have been conducted with Emx1-Cre; Nubp2flox/flox;p53/wt compound mutants to understand the interaction between NUBP2 deficiency and the p53 pathway .

These models allow researchers to assess the role of NUBP2 in specific tissues during development and to investigate the mechanisms underlying NUBP2-associated disorders.

How can neurosphere assays be used to evaluate NUBP2 function?

Neurosphere assays provide a valuable in vitro system for evaluating NUBP2 function and testing the pathogenicity of NUBP2 variants. The methodology involves:

• Isolation and culture: Cortical cells are isolated from embryonic mouse brains (typically at E12.5) and plated on low-adherence tissue culture plates. Since these cells are primarily progenitors, they cluster together and divide to form neurospheres .

• Experimental manipulations: Neurospheres can be derived from Nubp2-deficient models (e.g., Emx1-Cre/wt; Nubp2flox/flox) and compared to wild-type controls. Additionally, neurospheres can be transfected with various constructs, including wild-type NUBP2 and mutant variants .

• Analysis: The size of neurospheres is measured at different time points (e.g., 3, 5, and 7 days in culture). Significant differences in size between wild-type and Nubp2-deficient neurospheres indicate altered growth capacity .

• Rescue experiments: To confirm the specificity of NUBP2's role, rescue experiments can be performed by transfecting Nubp2-deficient neurospheres with wild-type NUBP2. The restoration of normal neurosphere size would confirm NUBP2's direct involvement in the observed phenotype .

• Variant testing: The functional impact of NUBP2 variants identified in patients can be assessed by expressing these variants in neurospheres and comparing their effects to those of wild-type NUBP2 .

In published research, wild-type neurospheres appeared much larger than Nubp2-deficient neurospheres, suggesting a reduced growth capacity in the absence of NUBP2. Transfection with wild-type NUBP2 rescued the size defect, confirming NUBP2's crucial role in neurosphere growth .

What techniques are effective for visualizing NUBP2 expression patterns?

Several techniques have proven effective for visualizing NUBP2 expression patterns:

• RNAScope RNA in situ hybridization: This technique has been successfully used to determine the expression pattern of Nubp2 in developing mouse brains. It allows for high-resolution visualization of mRNA distribution in tissues, enabling researchers to track expression changes across developmental stages and in specific cell types .

• Immunohistochemistry: While not explicitly mentioned in the provided search results, antibody-based detection of NUBP2 protein would complement RNA expression studies by revealing the protein's subcellular localization.

• Fluorescent protein tagging: For live cell imaging or subcellular localization studies, NUBP2 can be tagged with fluorescent proteins such as GFP and expressed in relevant cell types.

• Proximity ligation assays: These could be used to visualize and quantify NUBP2's interactions with known binding partners such as NUBP1 or other proteins it has been shown to interact with .

Each technique offers different advantages depending on the specific research question, with RNAScope providing excellent sensitivity for detecting gene expression in a spatial context within tissues.

How does NUBP2 deficiency contribute to primary microcephaly?

NUBP2 deficiency contributes to primary microcephaly through several interrelated mechanisms:

Animal models have confirmed that conditional deletion of Nubp2 from the mouse forebrain results in severe microcephaly, with significantly smaller forebrains and reduced cortical thickness. This phenotype closely resembles the primary microcephaly observed in human patients with NUBP2 variants .

What are the clinical features of patients with NUBP2 variants?

Patients with pathogenic NUBP2 variants display a constellation of clinical features, with primary microcephaly being the most prominent. Based on reported cases:

Family 1 Patient (Female):

• Severe intrauterine growth retardation (birth weight well below the 3rd percentile)

• Severe microcephaly (OFC 42.5 cm [-8SD], 43.5 cm [-7 to -8 SD], and 47 cm [-5SD] at three measurements between ages 15-20)

• Severe developmental delays and profound intellectual disability

• Absent speech and limited mobility

• Strabismus

• Short stature (height 130 cm [-5SD])

• Dysmorphic features including small cupped ears, arched eyebrows, upslanting palpebral fissures, and broad nasal bridge

Family 2 Patient (Fetus):

• Six-week developmental delay

• Head circumference and cerebellum below the 2nd centile

• Cervical kyphosis

• Micrognathia

• Short barrel-shaped chest

• Fixed, flexed elbows, fixed extended knees, clenched hands

• Ambiguous genitalia

The variant identified in Family 2 was a homozygous missense variant in NUBP2: NM_012225.4:c.334G>A: p.Ala112Thr. This variant segregated with the phenotype in an autosomal recessive manner and was predicted to be deleterious by multiple in silico tools (CADD=30, Polyphen=0.9, and SIFT=0.009) .

How do NUBP2 variants affect protein function at the molecular level?

The molecular effects of NUBP2 variants can be understood through several mechanisms:

• Disruption of protein-protein interactions: NUBP2 interacts with multiple partners, including NUBP1, with which it forms a functional complex. Variants may disrupt these interactions, impairing the protein's ability to participate in iron-sulfur cluster assembly or centrosome regulation .

• Impaired enzymatic activity: As an ATP-binding protein, NUBP2 likely has ATPase activity that could be compromised by certain variants, affecting its ability to participate in energy-dependent processes .

• Altered subcellular localization: Variants might prevent proper localization of NUBP2 to the basal body of the primary cilium or other cellular compartments where it functions .

• Destabilization of protein structure: Some variants could affect protein folding or stability, leading to reduced levels of functional NUBP2 protein.

Neurosphere assays have been used to experimentally assess the functional impact of NUBP2 variants. For example, expression of the NUBP2 Ala112Thr variant (identified in a patient with microcephaly) in neurospheres demonstrated functional differences compared to wild-type NUBP2, supporting its pathogenicity .

How does NUBP2 coordinate with NUBP1 in iron-sulfur cluster biogenesis?

The coordination between NUBP2 and NUBP1 in iron-sulfur cluster biogenesis represents a complex cellular process:

• Protein complex formation: NUBP2 and NUBP1 likely form a heterodimeric or multimeric complex that facilitates iron-sulfur cluster assembly. The specific stoichiometry and structural requirements for this complex formation remain areas for further investigation .

• Sequential steps in Fe-S cluster assembly: The NUBP1/NUBP2 complex appears to act upon iron and sulfur to create Fe4-S4 iron-sulfur clusters. These clusters serve as catalytic subunits in enzymes like aconitase, which are crucial for cellular metabolism. The precise mechanisms by which these proteins coordinate metal binding and transfer remain to be fully elucidated .

• Integration with other components: The cytosolic iron-sulfur cluster protein assembly pathway involves multiple components beyond NUBP1 and NUBP2. Understanding how these proteins coordinate with other pathway members, including those involved in iron sensing and transport, represents an important research direction .

• Regulatory mechanisms: How cells regulate the activity of NUBP1 and NUBP2 in response to changing iron levels or metabolic demands remains poorly understood and represents a critical area for future research.

Advanced techniques such as structural biology approaches (X-ray crystallography, cryo-EM), biochemical reconstitution, and real-time monitoring of Fe-S cluster assembly could help address these questions.

What is the relationship between NUBP2's roles in Fe-S cluster biogenesis and centrosome regulation?

The dual functionality of NUBP2 in Fe-S cluster biogenesis and centrosome regulation raises intriguing questions about potential mechanistic connections:

• Iron homeostasis and centrosome regulation: Loss of NUBP2 results in increased cytoplasmic iron, which may directly or indirectly affect centrosome dynamics. Research is needed to determine whether iron levels influence centrosome duplication or function .

• Shared protein interactions: NUBP2 may interact with proteins involved in both processes, suggesting a molecular bridge between iron-sulfur cluster biogenesis and centrosome regulation. Identifying these shared interactors could reveal novel regulatory mechanisms .

• Post-translational modifications: Fe-S clusters can function as cofactors that influence protein stability or activity. It's possible that Fe-S clusters mediate post-translational modifications of centrosomal proteins, connecting these two NUBP2 functions .

• Cell cycle coordination: Fe-S proteins are involved in DNA metabolism and repair, which are tightly coordinated with centrosome duplication during the cell cycle. NUBP2 might help synchronize these processes through its dual roles .

Experimental approaches to investigate these connections could include proximity labeling to identify proteins that interact with NUBP2 at centrosomes versus in the cytoplasm, and manipulating iron levels to assess effects on centrosome dynamics in wild-type versus NUBP2-deficient cells.

How might therapeutic approaches be developed for NUBP2-associated microcephaly?

Developing therapeutic approaches for NUBP2-associated microcephaly presents significant challenges but several potential strategies could be explored:

Research using the established mouse models and neurosphere assays would be invaluable for testing these potential therapeutic approaches before clinical translation .

What are the optimal experimental models for studying NUBP2 function in different contexts?

Different experimental models offer unique advantages for studying various aspects of NUBP2 function:

For studying NUBP2's role in neurodevelopment, conditional knockout mice and neurosphere assays have proven particularly valuable . For biochemical studies of iron-sulfur cluster assembly, cell-free systems or easily manipulated cell lines might be preferable. The choice of model should be guided by the specific research question and technical considerations.

How can researchers differentiate between NUBP2's direct effects and secondary consequences?

Differentiating between direct effects of NUBP2 deficiency and secondary consequences requires careful experimental design:

• Temporal analysis: Examining phenotypes at multiple time points can help establish the sequence of events. In the case of NUBP2-associated microcephaly, measuring brain size and cortical thickness at various developmental stages has revealed that differences become more pronounced over time, suggesting both primary and cumulative effects .

• Rescue experiments: Reintroducing wild-type NUBP2 into deficient systems provides strong evidence for direct effects. For example, transfection of Nubp2-deficient neurospheres with wild-type NUBP2 rescued their size defect, confirming NUBP2's direct role in neurosphere growth .

• Domain-specific mutations: Creating variants that selectively disrupt specific NUBP2 functions (e.g., iron-sulfur cluster assembly versus centrosome regulation) could help dissect which functions are responsible for particular phenotypes.

• Epistasis analysis: Manipulating downstream pathways (e.g., iron homeostasis, cell cycle regulators) in NUBP2-deficient models can reveal whether these pathways mediate NUBP2's effects. The creation of compound mutants, such as Emx1-Cre; Nubp2flox/flox;p53/wt, represents one approach to this type of analysis .

• Direct biochemical assays: Measuring iron-sulfur cluster formation, iron levels, or other biochemical parameters directly in NUBP2-deficient versus control cells can establish immediate consequences of NUBP2 loss.

These approaches, often used in combination, can help researchers build a mechanistic understanding of NUBP2's functions and their relationship to disease phenotypes.

What are the challenges in translating NUBP2 research from model systems to human patients?

Translating NUBP2 research from model systems to human patients faces several challenges:

• Species differences in neurodevelopment: While mouse models have been valuable for studying NUBP2 function, human brain development differs in timing, scale, and complexity. These differences may affect how NUBP2 deficiency manifests across species .

• Genetic background effects: The impact of NUBP2 variants may be modified by genetic background, both in model organisms and in humans. This variability complicates the interpretation of genotype-phenotype correlations.

• Severity and timing of deficiency: Complete knockout of NUBP2 in model systems may not accurately reflect the partial loss of function often seen with human variants. Additionally, the developmental timing of NUBP2 disruption may differ between experimental models and human patients .

• Limited patient cohorts: With only a few reported patients with NUBP2 variants, establishing a comprehensive clinical picture of NUBP2-associated disorders remains challenging. More patient identification and detailed phenotyping are needed .

• Prenatal onset: Since NUBP2-associated microcephaly begins prenatally, therapeutic intervention would need to occur during pregnancy, presenting significant practical and ethical challenges.

• Functional validation of variants: While neurosphere assays provide a valuable tool for assessing variant pathogenicity, the complexity of NUBP2's functions means that some variants may have subtle or context-specific effects that are difficult to model experimentally .

Addressing these challenges will require multidisciplinary approaches combining basic research, clinical genetics, and translational medicine to bridge the gap between model systems and human patients.

What are the most pressing unanswered questions in NUBP2 research?

Despite significant progress in understanding NUBP2's functions and disease associations, several critical questions remain:

• What is the complete spectrum of human phenotypes associated with NUBP2 variants? The identification of additional patients with diverse NUBP2 variants will help establish genotype-phenotype correlations and potentially reveal new disease associations beyond microcephaly .

• How do NUBP2's roles in iron-sulfur cluster assembly and centrosome regulation intersect mechanistically? Understanding potential crosstalk between these functions could reveal novel cellular regulatory mechanisms .

• What is the three-dimensional structure of NUBP2, both alone and in complex with NUBP1 or other interaction partners? Structural insights would facilitate a deeper understanding of how variants affect protein function and could guide therapeutic development.

• Are there tissue-specific requirements for NUBP2 beyond the brain? While neurological phenotypes are prominent in patients with NUBP2 variants, the protein may have important functions in other tissues that contribute to patient phenotypes or remain to be discovered .

• How do cells regulate NUBP2 activity in response to changing metabolic demands or stress conditions? Understanding the regulatory mechanisms controlling NUBP2 function could reveal new therapeutic targets.

Addressing these questions will require interdisciplinary approaches combining genetics, cell biology, biochemistry, and clinical research.

How might future technological advances enhance NUBP2 research?

Emerging technologies hold promise for advancing NUBP2 research in several areas:

• Single-cell technologies: Single-cell RNA sequencing and spatial transcriptomics could reveal cell type-specific requirements for NUBP2 and help identify the most vulnerable cell populations in NUBP2-deficient tissues .

• CRISPR-based approaches: Base editing and prime editing technologies could enable precise modeling of human NUBP2 variants in cellular and animal models, improving the translation between basic research and clinical applications.

• Advanced imaging: Super-resolution microscopy and live-cell imaging techniques could provide new insights into NUBP2's subcellular localization, dynamics, and interactions with centrosomes and other cellular structures.

• Proteomics advances: Proximity labeling and quantitative interaction proteomics could comprehensively map NUBP2's interactome in different cellular contexts, revealing new functional connections.

• Human brain organoids: Increasingly sophisticated brain organoid models derived from patient iPSCs could enable detailed studies of how NUBP2 variants affect human neurodevelopment in a more physiologically relevant context than current models .

These technological advances, combined with growing clinical awareness of NUBP2-associated disorders, are likely to accelerate progress in understanding NUBP2's functions and developing potential therapeutic approaches.

What collaborative approaches might accelerate progress in NUBP2 research and clinical translation?

Progress in NUBP2 research and its clinical translation could be accelerated through several collaborative approaches:

• Patient registries and consortia: International collaborations like the Care4Rare Canada program that identified NUBP2 variants demonstrate the value of sharing genetic and clinical data across institutions and countries . Expanding these efforts could identify more patients and establish comprehensive phenotypic spectra.

• Interdisciplinary research teams: Combining expertise from developmental biology, neuroscience, structural biology, and clinical genetics would provide complementary perspectives on NUBP2 function and pathogenicity.

• Shared model systems and reagents: Developing and sharing validated research tools—such as antibodies, cell lines, and animal models—would reduce redundant efforts and enable more rapid progress.

• Open science initiatives: Preregistration of studies, open access publication, and data sharing would maximize the impact of NUBP2 research and facilitate replication and extension of key findings.

• Industry-academic partnerships: Collaborations with pharmaceutical companies could accelerate the development of therapeutic approaches for NUBP2-associated disorders, particularly for approaches like small molecule screening that benefit from industry expertise and resources.