NUBP1 Human

What is NUBP1 and what protein family does it belong to?

NUBP1 is a nucleotide-binding protein that belongs to the MRP/MinD-type P-loop NTPases family with sequence similarity to bacterial division site-determining proteins. It is part of a highly conserved family of proteins found throughout eukaryotes . The NUBP/MRP gene family is well conserved throughout phylogeny, with NUBP1 representing the "long form" characterized by a unique N-terminal sequence containing four cysteine residues that is lacking in the related protein NUBP2 .

These proteins share conserved ATP/GTP-binding motifs (P-loop) and other highly conserved sequence motifs known as NUBP/MRP motifs alpha and beta . Interestingly, while prokaryotes generally possess only one type of NUBP/MRP gene, eukaryotes have evolved two distinct types, with NUBP1 being part of a group that includes human NBP, yeast NBP35, and C. elegans F10G8.6 .

What are the structural characteristics of human NUBP1?

Human NUBP1 is a 320-amino acid protein with several distinctive structural features . Its structure contains:

• An N-terminal CX₁₃CX₂CX₅C motif that serves as a binding site for a [4Fe-4S] cluster

• A C-terminal CPXC motif that also binds a [4Fe-4S] cluster

• Conserved ATP/GTP-binding motifs (P-loop and A')

The [4Fe-4S] clusters bound to these motifs show different stability characteristics:

• The N-terminal cluster binding site forms a stable association with [4Fe-4S] clusters

• The C-terminal cluster binding site shows greater lability, with clusters more easily lost over time

Spectroscopic analysis of NUBP1 reveals that the protein, when binding [4Fe-4S]²⁺ clusters, exhibits a broad absorption band at approximately 410 nm, characteristic of such clusters . When chemically reduced, NUBP1 produces distinctive EPR signals, further confirming the presence of these clusters .

What is the cellular localization of NUBP1?

NUBP1 shows specific subcellular localization patterns that provide insight into its functions:

• It is an integral component of centrioles throughout the cell cycle

• It localizes to the basal body of primary cilia in quiescent vertebrate cells

• It is present in invertebrate sensory cilia

• It can be found in motile cilia of mouse cells and in flagella

This localization pattern is independent of its interaction partner KIFC5A (a minus-end directed motor protein), suggesting that NUBP1 recruitment to these structures occurs through alternative mechanisms . The presence of NUBP1 at these key cellular structures aligns with its functional role in regulating centriole duplication and ciliary formation.

What is the relationship between NUBP1 and NUBP2?

NUBP1 and NUBP2 share evolutionary relationships but have distinct characteristics and functions:

• Both proteins belong to the NUBP/MRP gene family but represent different types in eukaryotes

• NUBP1 contains a unique N-terminal sequence with four cysteine residues that is absent in NUBP2

• The proteins physically interact with each other, as demonstrated by co-immunoprecipitation studies

• Despite their interaction, they have differential effects when individually knocked down:

  • NUBP1 silencing results in significant centrosome amplification and formation of multipolar spindles

  • NUBP2 appears to have a modulatory/accessory role for centriole arithmetic, working in concert with KIFC5A and NUBP1

In genomic terms, mouse Nubp2 is mapped to the t-complex region of mouse Chromosome 17, whereas Nubp1 is mapped to the proximal region of mouse Chromosome 16. Both regions are syntenic with human chromosome 16p13.1-p13.3, suggesting that a chromosomal breakage between these genes likely occurred during mouse chromosome evolution .

What role does NUBP1 play in Fe-S cluster assembly and transfer?

NUBP1 serves a crucial function in cellular Fe-S cluster biochemistry, acting as both a recipient of clusters and potentially as a scaffold for cluster assembly:

• Cluster Reception and Binding:
  NUBP1 can receive [2Fe-2S] clusters from the glutaredoxin protein GLRX3, which acts as a [2Fe-2S] cluster chaperone . When [2Fe-2S]₂-GLRX3₂-GS₄ is incubated with apo-NUBP1 under anaerobic conditions, cluster transfer occurs, as monitored by UV-vis spectroscopy, paramagnetic ¹H NMR, and iron/sulfide quantification .

• Differential Cluster Binding:
  NUBP1 contains two distinct cluster-binding sites:

  • The N-terminal CX₁₃CX₂CX₅C motif binds [4Fe-4S] clusters with high stability

  • The C-terminal CPXC motif also binds [4Fe-4S] clusters, but these show greater lability

• [4Fe-4S] Cluster Assembly:
  The experimental evidence suggests that NUBP1 may assemble [4Fe-4S] clusters from transferred [2Fe-2S] clusters. This is supported by spectroscopic changes observed when [2Fe-2S] clusters from GLRX3 are transferred to NUBP1 .

The methodological approach to study this process involves:

• Anaerobic protein preparation and handling

• Site-directed mutagenesis to create variants with specific cysteine motifs altered

• Spectroscopic techniques including UV-vis absorption and EPR

• Paramagnetic ¹H NMR to monitor cluster environment

• Quantitative iron and acid-labile sulfide assays

How does NUBP1 contribute to centriole function and regulation?

NUBP1 plays a critical role in centriole biology, serving as a negative regulator of centriole duplication:

• Integral Centriole Component:
  NUBP1 is present at centrioles throughout the cell cycle, suggesting a constitutive role in centriole structure or function .

• Regulation of Centriole Duplication:

  • Silencing of NUBP1 results in significant centriole amplification throughout the cell cycle

  • This leads to supernumerary centrosomes that can organize microtubule asters

  • The consequence is the formation of multipolar spindles during mitosis

• Mechanism of Action:
  The mechanism appears to involve:

  • Interaction with KIFC5A, a minus-end directed motor protein

  • Regulation of centrosome dynamics

  • Potential involvement in cytokinesis, as NUBP1 depletion partly causes cytokinesis defects

Experimental approaches to study NUBP1's role in centriole function include:

• siRNA-mediated knockdown in cultured cells

• Immunofluorescence microscopy to visualize centrosome number and spindle formation

• Cell cycle synchronization and analysis

• Flow cytometry to assess cell cycle distribution

• Quantitative Western blotting to monitor protein levels across the cell cycle

What are the phenotypic consequences of NUBP1 mutation or depletion?

NUBP1 dysfunction leads to diverse phenotypic consequences across multiple biological systems:

In mouse models (Nubp1ᵐ¹ᴺⁱˢʷ mutants):

• Developmental abnormalities including:

  • Syndactyly (fused digits)

  • Eye cataracts

  • Lung hypoplasia

• Lung-specific effects:

  • Increased apoptosis in lung tissue

  • Loss of distal progenitor markers including Sftpc, Sox9, and Foxp2

  • Disrupted localization of polarity protein Par3 and mitosis-relevant protein Numb

In cellular models:

• Centrosome abnormalities:

  • Supernumerary centrosomes

  • Multipolar spindles

  • Aberrant microtubule organization

• Cell division defects:

  • Cytokinesis failures leading to multinucleated cells

  • Genomic instability due to improper chromosome segregation

These phenotypes highlight NUBP1's essential roles in:

• Developmental morphogenesis

• Cellular polarity

• Centrosomal dynamics

• Progenitor cell survival and function

The diverse phenotypic consequences suggest that NUBP1 functions at the intersection of multiple cellular processes, with its dysfunction having pleiotropic effects depending on the cellular and developmental context.

What methodologies are most effective for studying NUBP1 function in cell culture?

Several complementary methodological approaches have proven effective for investigating NUBP1 function:

• Gene Silencing Techniques:

  • siRNA-mediated knockdown allows targeted depletion of NUBP1

  • Real-time RT-PCR to confirm knockdown efficiency using primers specific for NUBP1

  • Comparison with related proteins (NUBP2, KIFC5A) to distinguish specific vs. shared functions

• Protein-Protein Interaction Studies:

  • Co-immunoprecipitation to identify interaction partners

  • Proximity labeling approaches to map the NUBP1 interactome

  • Yeast two-hybrid screening for novel interactors

• Cell Cycle Analysis:

  • Thymidine block synchronization to study NUBP1 function across cell cycle phases

  • Flow cytometry with propidium iodide staining to quantify cell cycle distribution

  • Quantitative Western blotting to monitor NUBP1 protein levels throughout the cell cycle

• Microscopy Techniques:

  • Immunofluorescence to visualize NUBP1 localization and centrosome/centriole numbers

  • Live-cell imaging to track centrosome dynamics in NUBP1-depleted cells

  • Super-resolution microscopy for detailed structural analysis

• Biochemical Approaches for Fe-S Cluster Studies:

  • Anaerobic protein purification to maintain cluster integrity

  • UV-vis spectroscopy to monitor cluster binding

  • EPR and paramagnetic ¹H NMR for detailed cluster characterization

  • Mutagenesis of cysteine motifs to study specific cluster-binding sites

Statistical analysis of experimental data typically employs:

• Student's t-tests for comparing two conditions

• ANOVA with appropriate post-tests (Tukey's, Bonferroni) for multiple comparisons

• Mean ± standard deviation from at least three independent experiments

How can researchers distinguish between the functions of NUBP1's N-terminal and C-terminal motifs?

Distinguishing the functions of NUBP1's distinct domains requires targeted experimental approaches:

• Domain-Specific Mutants:
  Researchers have successfully employed two key mutant constructs:

  • NUBP1³⁸⁻³²⁰: A truncated variant lacking the N-terminal CX₁₃CX₂CX₅C motif, retaining only the C-terminal CPXC motif

  • NUBP1-C235A/C238A: A full-length variant with the two cysteines of the CPXC motif mutated to alanines, thus inactivating the C-terminal motif while preserving the N-terminal motif

• Spectroscopic Differentiation:
  These mutants show distinctive spectroscopic signatures:

  • UV-vis spectra of both mutants exhibit absorption bands at ~410 nm, characteristic of [4Fe-4S]²⁺ clusters

  • EPR signals differ between clusters bound at N-terminal versus C-terminal sites

  • Paramagnetic ¹H NMR reveals differential hyperfine shifted signals

• Cluster Stability Assessment:
  Experimental approaches to assess differential cluster stability include:

  • Time-course monitoring of UV-vis spectra under anaerobic conditions

  • Serial acquisition of paramagnetic ¹H NMR spectra to track signal degradation

  • These reveal that C-terminal bound clusters are significantly more labile than N-terminal bound clusters

• Functional Transfer Experiments:
  Incubating [2Fe-2S]-loaded GLRX3 with domain-specific NUBP1 mutants allows researchers to determine:

  • Which domain preferentially receives clusters

  • Whether specific domains are involved in cluster conversion ([2Fe-2S] to [4Fe-4S])

  • The stoichiometry of cluster transfer to each domain

These approaches have revealed important functional differences: