UBA2 Human

What is UBA2 and what is its primary function in human cells?

UBA2 (ubiquitin-like modifier activating enzyme 2) is a crucial enzyme involved in the SUMOylation pathway, a post-translational modification process. Unlike ubiquitination, SUMOylation does not primarily target proteins for degradation, but instead regulates cell cycle progression, subcellular trafficking, signal transduction, stress responses, and chromatin structure dynamics . UBA2 forms a heterodimer with SAE1 (SUMO-Activating Enzyme Subunit 1) and binds with SUMO1 in an ATP-dependent manner, acting as the E1-activating enzyme that initiates the SUMOylation cascade . This process is fundamental to maintaining transcriptional regulation of tissue-specific gene expression through the modification of protein kinases and transcription factors .

What is the genomic location and structure of the UBA2 gene?

The UBA2 gene is located on chromosome 19, specifically at chromosomal band 19q13.11 in humans . It is also known by several synonyms including ARX, HRIHFB2115, and SAE2 . The gene encodes a protein-coding transcript that plays a critical role in the SUMOylation pathway. UBA2 lies adjacent to the minimal deletion overlap region associated with chromosome 19q13.11 deletion syndrome . This genomic positioning is significant for understanding both isolated UBA2 variants and larger chromosomal aberrations affecting this region.

How does UBA2 contribute to cellular homeostasis through SUMOylation?

UBA2's role in SUMOylation represents a sophisticated regulatory mechanism for cellular homeostasis. As part of the E1-activating enzyme complex with SAE1, UBA2 initiates the SUMOylation process by activating SUMO proteins in an ATP-dependent manner . This activation enables the subsequent transfer of SUMO to target proteins, modifying their function, localization, or interactions. SUMOylation regulated by UBA2 affects:

• Cell cycle regulation

• Nuclear-cytoplasmic transport

• Protein stability and function

• Transcriptional regulation

• DNA damage responses

• Stress responses

Through these mechanisms, UBA2 maintains cellular homeostasis and enables appropriate cellular responses to environmental changes and stressors .

What is the evolutionary conservation of UBA2 across species?

UBA2 shows significant evolutionary conservation across species, indicating its fundamental importance in cellular processes. The zebrafish homolog (uba2) has been extensively studied as a model for human UBA2 function . Research has demonstrated that uba2 is expressed in zebrafish eye, brain, and pectoral fins during development . The functional conservation between human UBA2 and zebrafish uba2 is so substantial that human UBA2 mRNA can be used in rescue experiments in uba2-null zebrafish . This evolutionary conservation makes animal models particularly valuable for studying the effects of UBA2 variants identified in human patients.

What syndrome is associated with UBA2 variants and what is its phenotypic spectrum?

UBA2 variants cause a recognizable syndrome with a wide phenotypic spectrum. Clinical features include:

• Aplasia cutis congenita (ACC)

• Ectrodactyly/oligodactyly

• Neurodevelopmental abnormalities

• Ectodermal variations

• Skeletal anomalies

• Craniofacial abnormalities

• Cardiac defects

• Renal anomalies

• Genital anomalies

• Ocular abnormalities

Both loss-of-function and missense sequence variants in UBA2 have been identified in affected individuals. The syndrome bears similarities to features seen in chromosome 19q13.11 deletion syndrome, confirming UBA2's causal role in the developmental phenotypes associated with this chromosomal deletion .

How do UBA2 mutations impact development in model organisms?

In zebrafish models, loss of uba2 function results in multiple developmental abnormalities that parallel human phenotypes:

• Deficient growth

• Microcephaly

• Microphthalmia (small eyes)

• Mandibular hypoplasia

• Abnormal fin development

• Mild to severe hydrocephaly

• Uninflated swim bladder

These developmental impacts suggest critical roles for UBA2 in craniofacial development, brain formation, and limb/fin development. The zebrafish model has been particularly valuable because uba2 expression patterns in developing zebrafish correlate with the tissues affected in human patients with UBA2 variants. Expression is notably detected in the eye, brain, and pectoral fins during crucial developmental periods .

What is the role of UBA2 in cancer, particularly in gliomas?

Recent research has identified UBA2 as a potential prognostic biomarker and therapeutic target in glioma. Studies have found:

• UBA2 is overexpressed in glioma tissues and cell lines compared to normal tissues

• UBA2 expression correlates with World Health Organization (WHO) grade, IDH gene status, 1p19q deletion, and histological features in gliomas

• UBA2 has demonstrated diagnostic and prognostic value in glioma

• UBA2 may be associated with tumor progression and immune cell responses in the tumor microenvironment

These findings suggest that UBA2 may play a significant role in glioma development and progression, potentially through dysregulation of SUMOylation of key proteins involved in cell proliferation, survival, and immune evasion.

How do different types of UBA2 variants affect protein function and phenotype?

UBA2 variants exhibit variable effects on protein function, leading to a spectrum of phenotypic manifestations. Two primary categories have been identified:

• Loss-of-function variants: These typically cause more severe phenotypes by significantly reducing or eliminating UBA2 activity in the SUMOylation pathway .

• Missense variants: These may have more variable effects depending on the specific amino acid change and its location within functional domains of the protein .

Zebrafish rescue experiments have been instrumental in classifying these variants. When human UBA2 mRNA containing identified missense variants was injected into uba2-null zebrafish, it failed to rescue the mutant phenotype, confirming the pathogenicity of these variants . This functional evidence supports that both complete loss of function and specific missense changes can disrupt UBA2's role in the SUMOylation pathway, leading to developmental abnormalities.

What animal models are most effective for studying UBA2 function?

Zebrafish have emerged as the preferred animal model for studying UBA2 function due to several advantages:

• Evolutionary conservation: The zebrafish uba2 gene shows significant homology to human UBA2, enabling translational insights .

• Developmental accessibility: Transparent embryos allow for real-time visualization of developmental processes affected by uba2 disruption .

• Genetic manipulation ease: CRISPR/Cas9 techniques have been successfully used to generate uba2 knockout lines .

• Expression pattern relevance: uba2 is expressed in zebrafish tissues (eye, brain, pectoral fins) that correspond to affected systems in human patients .

• Rescue experiment feasibility: Human UBA2 mRNA can be injected into uba2-null zebrafish to assess variant pathogenicity .

The methodology for establishing zebrafish uba2 models typically involves:

• CRISPR/Cas9-mediated knockout of uba2

• Whole mount in situ hybridization to characterize expression patterns

• Phenotypic analysis at multiple developmental stages (24hpf, 48hpf, 72hpf, 5dpf, 7dpf)

• Rescue experiments with wild-type or variant human UBA2 mRNA

How can researchers validate the pathogenicity of novel UBA2 variants?

A multi-faceted approach is recommended for validating the pathogenicity of novel UBA2 variants:

• Bioinformatic prediction: Assess conservation, structural impacts, and predicted functional effects using algorithms like SIFT, PolyPhen, and CADD.

• Segregation analysis: Determine if the variant segregates with disease in affected families.

• Functional assays in cellular models:

  • Measure SUMOylation efficiency in cells expressing variant UBA2

  • Assess heterodimer formation with SAE1

  • Evaluate ATP binding and SUMO activation

• Zebrafish rescue experiments:

  • Generate uba2-null zebrafish

  • Inject human UBA2 mRNA (wild-type and variant forms)

  • Compare rescue efficiency between wild-type and variant UBA2

This approach has successfully demonstrated that human UBA2 mRNAs containing missense variants failed to rescue nullizygous zebrafish phenotypes, providing strong evidence for pathogenicity .

What techniques are most effective for assessing UBA2 expression in tissues?

Multiple complementary techniques have proven effective for assessing UBA2 expression in various tissues:

• In situ hybridization (ISH):

  • Whole mount ISH in developing embryos at multiple developmental stages (5 somite, 24hpf, 35hpf, 48hpf, 72hpf, 5dpf, 7dpf)

  • Section ISH for precise localization in specific tissue layers

• Immunohistochemistry (IHC):

  • Antibody-based detection of UBA2 protein in tissue sections

  • Dual staining with tissue-specific markers for co-localization studies

• Quantitative PCR (qPCR):

  • Precise quantification of UBA2 mRNA expression levels

  • Comparison between normal and pathological tissues (especially relevant in glioma studies)

• Western blotting:

  • Assessment of UBA2 protein levels

  • Detection of SUMOylated target proteins to assess pathway activity

• Bioinformatic analysis using public databases:

  • Mining expression data from cancer databases and normal tissue atlases

  • Correlation of expression with clinical parameters and outcomes

What are the best methods for studying UBA2-mediated SUMOylation in experimental settings?

To effectively study UBA2-mediated SUMOylation, researchers should consider these methodological approaches:

• In vitro SUMOylation assays:

  • Purified recombinant UBA2 and SAE1 proteins

  • ATP-dependent activation of SUMO

  • Detection of SUMOylated substrate proteins by Western blot

• Cell-based SUMOylation reporter systems:

  • Fluorescent or luminescent reporters fused to SUMO substrates

  • Real-time monitoring of SUMOylation in living cells

  • High-throughput screening capabilities

• Proximity ligation assays:

  • Detection of UBA2-SAE1 interactions in situ

  • Visualization of protein-protein interactions within cellular compartments

• Proteomic approaches:

  • Mass spectrometry to identify SUMOylation targets

  • Quantitative proteomics to measure changes in the SUMOylation profile following UBA2 manipulation

• CRISPR/Cas9-mediated gene editing:

  • Generation of UBA2 knockout or knockin cell lines

  • Introduction of specific variants identified in patients

  • Assessment of global SUMOylation changes

These methods collectively provide a comprehensive toolbox for investigating UBA2 function in the SUMOylation pathway and understanding how variants disrupt this critical cellular process.

How does UBA2 expression correlate with drug sensitivity in cancer?

Research on UBA2 in glioma has begun exploring the correlation between UBA2 expression and drug sensitivity in cancer treatments . While specific data from the search results is limited, the general research approach includes:

• Bioinformatic analysis of UBA2 expression in relation to drug response data from cancer databases

• Cell line experiments examining differential drug sensitivity in cells with various UBA2 expression levels

• Assessment of SUMOylation status of drug targets and resistance factors

This research direction is particularly promising because SUMOylation affects multiple proteins involved in drug resistance mechanisms, including DNA repair proteins, multidrug resistance transporters, and anti-apoptotic factors. Targeting UBA2 could potentially sensitize cancer cells to existing chemotherapeutics by modulating these resistance pathways.

What is the relationship between UBA2 dysregulation and immune responses in the tumor microenvironment?

The relationship between UBA2 and immune responses in the tumor microenvironment represents an emerging research area, particularly in glioma. Initial findings suggest:

• UBA2 expression may be associated with immune cell infiltration and function in glioma

• SUMOylation can affect the expression of immune checkpoint molecules and cytokines

• UBA2-mediated modifications may influence tumor cell recognition by immune cells

This relationship is likely mediated through SUMOylation of transcription factors that regulate immune-related genes and signaling molecules. Research methodologies to investigate this connection include:

• Correlation analysis between UBA2 expression and immune cell signatures in tumor transcriptome data

• Flow cytometry characterization of tumor-infiltrating immune cells in UBA2-high versus UBA2-low tumors

• Assessment of immune checkpoint molecule expression following UBA2 manipulation

How do tissue-specific SUMOylation patterns mediated by UBA2 contribute to developmental phenotypes?

The diverse developmental phenotypes observed in UBA2-related disorders likely result from tissue-specific SUMOylation patterns during embryonic development. Advanced research in this area involves:

• Temporal and spatial mapping of SUMOylation targets during embryonic development

• Identification of tissue-specific SUMOylation substrates in tissues affected by UBA2 variants (skin, limbs, brain, heart)

• Assessment of transcription factor SUMOylation in tissue-specific progenitor cells

In zebrafish models, uba2 shows specific expression patterns in the eye, brain, and pectoral fins, correlating with the phenotypes observed in uba2-null fish (microphthalmia, microcephaly, and fin abnormalities) . This suggests that UBA2 regulates the SUMOylation of critical developmental factors in a tissue-specific manner. Understanding these tissue-specific patterns may explain the variable expressivity observed in human patients with UBA2 variants.

What are the molecular mechanisms underlying the relationship between UBA2 and chromosome 19q13.11 deletion syndrome?

The relationship between UBA2 and chromosome 19q13.11 deletion syndrome reveals complex genomic mechanisms:

• UBA2 lies adjacent to the minimal deletion overlap region in 19q13.11 deletion syndrome

• Isolated UBA2 variants produce phenotypes that overlap with features of the deletion syndrome

• Loss of UBA2 function appears to be a major driver of the developmental abnormalities in both conditions

Research approaches to further elucidate this relationship include:

• Detailed genotype-phenotype correlation studies in patients with deletions of various sizes

• Comparison of developmental outcomes in UBA2 point mutation cases versus deletion cases

• Investigation of potential position effects on UBA2 expression in deletion cases

• Examination of possible synergistic effects between UBA2 and neighboring genes

This research provides insight into how single-gene disorders relate to contiguous gene syndromes and helps prioritize candidate genes in chromosomal disorders.

How might UBA2-targeted therapeutics be developed for UBA2-associated disorders or cancers?

Development of UBA2-targeted therapeutics represents an emerging frontier with several potential strategies:

• Small molecule inhibitors of the UBA2-SAE1 heterodimer:

  • Target ATP binding site

  • Disrupt UBA2-SAE1 interaction

  • Block SUMO transfer

• Antisense oligonucleotides for UBA2 downregulation in cancers with UBA2 overexpression

• Gene therapy approaches for UBA2-deficient developmental disorders:

  • Delivery of functional UBA2 to affected tissues during development

  • Temporal and tissue-specific expression control

• Synthetic bypasses of the SUMOylation pathway for specific substrates affected by UBA2 deficiency

The differential requirements for UBA2 in normal versus cancer tissues provide a potential therapeutic window, particularly in gliomas where UBA2 overexpression has been observed . For developmental disorders, the challenge lies in delivering intervention early enough to prevent irreversible developmental abnormalities.

What controls should be included when studying UBA2 variants in functional assays?

Robust experimental design for studying UBA2 variants requires comprehensive controls:

• Positive controls:

  • Wild-type UBA2 expression constructs

  • Known functional UBA2 interactions (e.g., with SAE1)

  • Established SUMOylation substrates

• Negative controls:

  • Empty vector controls

  • Known non-functional UBA2 mutants (catalytic site mutations)

  • Non-SUMOylatable substrate mutants

• Variant-specific controls:

  • Conservative amino acid substitutions at the variant position

  • Variants classified as benign in population databases

  • Unrelated variants in the same protein domain

• Rescue experiment controls:

  • Dose-response titration of wild-type UBA2 mRNA in rescue experiments

  • Injection controls (vehicle-only)

  • Non-complementing gene mRNA injections

These controls help establish the specificity of observed effects and distinguish pathogenic from benign variation in UBA2.

How should researchers approach contradictory findings about UBA2 function across different model systems?

When facing contradictory findings about UBA2 function across different model systems, researchers should:

• Consider species-specific differences:

  • Evolutionary divergence in UBA2 structure or regulation

  • Differences in developmental timing or tissue-specific expression

  • Variation in SUMOylation substrates between species

• Evaluate methodological differences:

  • Complete knockout versus knockdown approaches

  • Acute versus chronic loss of function

  • Different timepoints of analysis

  • Methodological limitations of each system

• Employ integrative approaches:

  • Multiple model systems in parallel (cell lines, zebrafish, mouse models)

  • Complementary techniques to assess UBA2 function

  • Direct comparison using standardized assays

• Focus on human-relevant outcomes:

  • Prioritize findings that explain human phenotypes

  • Validate model system findings with patient samples when possible

  • Consider developmental context and timing

What statistical approaches are most appropriate for analyzing UBA2 expression data in relation to clinical outcomes?

When analyzing UBA2 expression data in relation to clinical outcomes, particularly in cancer studies, these statistical approaches are recommended:

• Survival analysis:

  • Kaplan-Meier analysis with log-rank test for comparing high versus low UBA2 expression groups

  • Cox proportional hazards regression for multivariate analysis incorporating UBA2 expression and other clinical variables

• Correlation analysis:

  • Spearman or Pearson correlation between UBA2 expression and continuous clinical variables

  • Point-biserial correlation for dichotomous outcomes

• Receiver Operating Characteristic (ROC) curve analysis:

  • Determine optimal UBA2 expression cutoffs for prognostic classification

  • Calculate area under the curve (AUC) to assess diagnostic/prognostic value

• Linear mixed models:

  • Account for repeated measurements and hierarchical data structures

  • Incorporate random effects to address inter-patient variability

• Multiple testing correction:

  • Benjamini-Hochberg procedure for controlling false discovery rate

  • Bonferroni correction for family-wise error rate in multiple comparisons

These approaches help establish the clinical relevance of UBA2 expression patterns while accounting for confounding factors and avoiding spurious associations.

How can researchers distinguish primary effects of UBA2 dysfunction from secondary consequences?

Distinguishing primary from secondary effects of UBA2 dysfunction requires sophisticated experimental design:

• Temporal analysis:

  • Time-course experiments following UBA2 manipulation

  • Identification of earliest molecular changes

  • Conditional/inducible systems for temporal control of UBA2 disruption

• Direct substrate analysis:

  • Identification of direct SUMOylation targets using proteomics

  • Verification with in vitro SUMOylation assays

  • Site-specific mutagenesis of SUMOylation sites in target proteins

• Rescue experiments with specificity controls:

  • Structure-function analysis with UBA2 domain mutants

  • Substrate-specific rescue approaches

  • Bypass of UBA2 requirement through direct expression of SUMO-substrate fusions

• Pathway analysis:

  • Systems biology approaches to model pathway relationships

  • Network analysis to distinguish primary nodes from downstream effects

  • Identification of feedback loops that may amplify initial perturbations

• Single-cell approaches:

  • Single-cell transcriptomics to identify cell type-specific responses

  • Trajectory analysis to map developmental consequences of UBA2 dysfunction

  • Spatial transcriptomics to localize primary effects within tissues

These approaches collectively enable researchers to trace the causal chain from UBA2 dysfunction through immediate biochemical consequences to ultimate phenotypic manifestations.

What considerations are important when designing rescue experiments for UBA2 variants?

When designing rescue experiments to evaluate UBA2 variants, particularly in zebrafish models, several critical considerations should be addressed:

• mRNA quality and dosage:

  • Use capped, polyadenylated mRNA for stability

  • Titrate injection amounts to determine optimal dosage

  • Verify translation using tagged constructs

• Timing of intervention:

  • Inject at early embryonic stages before endogenous expression begins

  • Consider critical developmental windows for affected tissues

  • Evaluate multiple developmental timepoints for rescue assessment

• Quantitative phenotype assessment:

  • Develop objective, quantifiable metrics for phenotypic rescue

  • Use imaging-based approaches for morphological features

  • Include functional assays relevant to the phenotype

• Controls and comparisons:

  • Include wild-type mRNA as positive control

  • Use known null variants as negative controls

  • Test multiple variants in parallel with standardized conditions

• Statistical power:

  • Ensure sufficient sample size for statistical significance

  • Account for embryonic lethality and developmental variability

  • Consider replicate experiments under identical conditions

The zebrafish model has been successfully used to demonstrate that human UBA2 mRNAs containing missense variants failed to rescue nullizygous zebrafish phenotypes, confirming the causality and pathogenicity of these variants . This approach provides a powerful system for functional evaluation of variants identified in human patients.