Recombinant Danio rerio Ufm1-specific protease 2 (ufsp2)

What is Ufm1-specific protease 2 (ufsp2) and what is its primary function in zebrafish?

Ufm1-specific protease 2 (ufsp2) is a crucial enzyme involved in the ubiquitin-fold modifier 1 (Ufm1) cascade in zebrafish. Its primary function is to activate Ufm1 by proteolytic processing, exposing a C-terminal glycine residue necessary for subsequent conjugation steps. In the Ufm1 modification pathway, activated Ufm1 reacts with Uba5 (E1-like enzyme) and is transferred to Ufc1 (E2-like enzyme) before being transferred to its target protein by Ufl1 (E3-like enzyme) . This post-translational modification system plays critical roles in various cellular processes including endoplasmic reticulum (ER) stress responses and telomere maintenance in zebrafish development .

How conserved is ufsp2 across species?

The ufsp2 gene is remarkably conserved across multiple species. Alignment studies of UFSP2 protein sequences from various multicellular organisms, including both plants and animals, have demonstrated that certain critical residues such as p.Tyr290 (in human UFSP2) have been conserved for at least 1.6 billion years . This extraordinary evolutionary conservation underscores the fundamental importance of UFSP2's function in cellular processes across diverse taxa. The high degree of conservation makes zebrafish an excellent model organism for studying the functions of ufsp2 that might be relevant to human health and disease.

What developmental processes in zebrafish are affected by ufsp2 function?

The UFMylation pathway, in which ufsp2 plays a key role, significantly impacts several developmental processes in zebrafish:

How does ufsp2 contribute to telomere maintenance in zebrafish, and what are the molecular mechanisms involved?

The contribution of ufsp2 to telomere maintenance involves several molecular mechanisms centered around MRE11 UFMylation:

• MRE11 UFMylation pathway: ufsp2 activates Ufm1, which is essential for the UFMylation of MRE11, a component of the MRN complex that plays a critical role in DNA repair and telomere maintenance .

• Phosphatase recruitment: UFMylation of MRE11 is necessary for the recruitment of phosphatase PP1-α, which leads to the dephosphorylation of NBS1 (another component of the MRN complex) .

• MRN complex recruitment: Without proper UFMylation, NBS1 remains phosphorylated, reducing MRN recruitment to telomeres .

• Telomere stability: The absence of MRN at telomeres favors the formation of the TRF2-Apollo/SNM1 complex, which is associated with the loss of leading telomeres .

• Functional evidence: Zebrafish expressing Mre11 that cannot be UFMylated phenocopy Ufm1-deficient zebrafish, demonstrating that UFMylation of MRE11 is an evolutionarily conserved mechanism regulating telomere length .

What is the relationship between ufsp2 dysfunction and premature aging in zebrafish models?

The relationship between ufsp2 dysfunction and premature aging in zebrafish appears to be primarily mediated through telomere maintenance:

• Telomere shortening: Disruption of the UFMylation pathway leads to telomere attrition in zebrafish, particularly evident in erythrocytes .

• Stem cell impairment: Telomere shortening in hematopoietic stem and progenitor cells (HSPCs) likely contributes to the observed anemia and reduced HSPC numbers in UFMylation-deficient zebrafish .

• Synergistic effects with telomerase deficiency: Genetic inactivation of components of the UFMylation pathway in telomerase-deficient zebrafish results in severely impaired development and drastically reduced lifespan, suggesting that UFMylation and telomerase pathways contribute to telomere maintenance through distinct but complementary mechanisms .

• Developmental consequences: Mre11a-deficient zebrafish, like Ufm1- and Ufl1-deficient zebrafish, show reduced lifespan and impaired development observable from approximately 1 month onward .

How do mutations in ufsp2 affect its proteolytic activity and subsequent cellular pathways?

Mutations in ufsp2 can significantly disrupt its proteolytic activity with cascading effects on downstream cellular pathways:

• Active site disruption: The p.Tyr290His mutation in human UFSP2 (corresponding to p.Tyr282His in mouse) inactivates UFSP2 proteolytic function. This tyrosine residue is crucial within the UFSP2 active site according to crystal structure predictions .

• Ufm1 processing impairment: In vitro assays with purified mutant mouse UFSP2 containing the equivalent mutation showed that it could not cleave Ufm1, even at high concentrations .

• Dominant-negative effects: Interestingly, in vitro studies suggest that mutated UFSP2 does not exert a dominant-negative effect at the level of Ufm1 processing, as the processing activity of wild-type UFSP2 was not affected in the presence of increasing concentrations of mutant UFSP2 .

• Downstream pathway disruption: Failure to activate Ufm1 properly would impact all subsequent UFMylation events, including the critical UFMylation of MRE11 necessary for telomere maintenance .

What are the recommended methods for generating recombinant Danio rerio ufsp2 for in vitro studies?

For generating recombinant Danio rerio ufsp2 for in vitro studies, the following methodological approach is recommended:

• Expression construct design:

  • Clone the full-length zebrafish ufsp2 cDNA into a suitable expression vector (e.g., pGEX for GST-tagged proteins)

  • Include appropriate affinity tags (such as GST, His, or HA) to facilitate purification

  • For mutation studies, site-directed mutagenesis can be performed on the wild-type construct

• Expression system:

  • Escherichia coli (E. coli) has been successfully used for expressing functional UFSP2 proteins

  • BL21(DE3) or similar strains are recommended for protein expression

• Protein purification:

  • Use affinity chromatography based on the chosen tag (e.g., glutathione-sepharose for GST-tagged proteins)

  • Include protease inhibitors during lysis to prevent degradation

  • Consider size-exclusion chromatography as a second purification step to ensure high purity

• Activity assay:

  • Assess proteolytic activity using tagged Ufm1 substrates (e.g., GST-Ufm1-HA)

  • Analyze cleavage products by SDS-PAGE gel electrophoresis

  • Visualize protein bands with Coomassie blue staining or western blotting

What are the effective approaches for genetic manipulation of ufsp2 in zebrafish models?

Effective approaches for genetic manipulation of ufsp2 in zebrafish include:

• CRISPR-Cas9 gene editing:

  • Design guide RNAs targeting specific regions of the ufsp2 gene

  • Inject CRISPR-Cas9 components into one-cell stage zebrafish embryos

  • Validate editing efficiency through sequencing and/or restriction enzyme digestion

• Transgenic approaches:

  • Use fluorescent reporter lines (such as Tg(lcr:eGFP) for erythroid cells, Tg(runx1:nfsB-mCherry) for HSPCs, or Tg(mpx:eGFP) for neutrophils) to monitor phenotypic effects

  • These reporter lines allow for visualization and quantification of specific cell populations affected by ufsp2 manipulation

• Rescue experiments:

  • Perform mRNA injection of wild-type or mutant ufsp2 into ufsp2-deficient zebrafish embryos

  • These experiments can confirm the specificity of observed phenotypes and test the functional consequences of specific mutations

• Generation of stable mutant lines:

  • Establish F0 mosaic mutants through CRISPR-Cas9

  • Breed these fish to generate stable F1 heterozygotes and eventually F2 homozygous mutants

  • Characterize the resulting mutant lines through molecular and phenotypic analyses

What techniques are most reliable for assessing UFMylation activity in zebrafish tissues?

For assessing UFMylation activity in zebrafish tissues, the following techniques have proven reliable:

• Telomere length analysis:

  • Quantitative PCR (qPCR) can be used to measure relative telomere length in whole zebrafish larvae or specific cell populations

  • Quantitative fluorescence in situ hybridization (qFISH) offers an alternative approach for measuring telomere length in specific cell types (e.g., erythrocytes)

• Protein interaction studies:

  • Co-immunoprecipitation assays to detect interactions between UFMylation pathway components

  • Pull-down assays using tagged recombinant proteins to identify UFMylation substrates

• Cell-specific analyses:

  • Fluorescence-activated cell sorting (FACS) of specific cell populations (e.g., erythrocytes or HSPCs) from transgenic reporter lines

  • This approach allows for the analysis of UFMylation activity in specific cell types rather than whole larvae

• Phenotypic assessment:

  • Monitoring developmental milestones and survival rates

  • Quantification of specific cell populations (e.g., erythrocytes, HSPCs, neutrophils) using transgenic reporter lines

  • These assessments can serve as functional readouts of UFMylation pathway activity

What are the main technical challenges in studying recombinant ufsp2 function in zebrafish models?

Several technical challenges exist when studying recombinant ufsp2 function in zebrafish:

• Tissue-specific expression patterns:

  • The complete tissue-specific expression pattern of ufsp2 during zebrafish development remains incompletely characterized

  • This complicates the interpretation of phenotypes observed in ufsp2-deficient models

• Functional redundancy:

  • The potential functional overlap between UFSP1 and UFSP2 in zebrafish must be considered

  • Both proteases can activate Ufm1, potentially masking phenotypes in single protease knockouts

• Target identification:

  • The full repertoire of Ufm1 target proteins remains to be identified

  • This knowledge gap limits our understanding of the downstream effects of ufsp2 dysfunction

• Phenotypic analysis in larvae:

  • Technical challenges exist in analyzing telomere length in specific cell populations (like HSPCs) from zebrafish larvae due to low cell numbers

  • This has necessitated the use of erythrocytes as a surrogate for HSPCs in some studies

How can researchers distinguish between direct effects of ufsp2 manipulation and secondary consequences in zebrafish models?

To distinguish between direct effects of ufsp2 manipulation and secondary consequences:

• Temporal analysis:

  • Perform detailed time-course studies to identify the earliest detectable changes following ufsp2 disruption

  • Early effects are more likely to represent direct consequences of ufsp2 dysfunction

• Substrate-specific approaches:

  • Generate zebrafish expressing specific UFMylation substrates (e.g., MRE11) that cannot be UFMylated

  • Compare these phenotypes with those of ufsp2-deficient zebrafish to identify substrate-specific effects

• Rescue experiments:

  • Perform rescue experiments with wild-type ufsp2 or specific UFMylation substrates

  • The ability of a substrate to rescue specific aspects of the ufsp2-deficient phenotype would suggest a direct relationship

• Combined genetic models:

  • Create and analyze double mutants (e.g., ufsp2 and telomerase deficiency)

  • Synergistic effects may reveal distinct pathways affected by ufsp2

What is the current understanding of ufsp2's role in disease models beyond developmental contexts?

The current understanding of ufsp2's role in disease models beyond developmental contexts is still emerging:

• Beukes hip dysplasia (BHD):

  • A mutation in UFSP2 (c.868T>C, p.Tyr290His) has been identified in an extended South African family with BHD

  • This rare autosomal dominant condition is characterized by severe progressive degenerative osteoarthritis of the hip joint in early adulthood

  • The mutation inactivates UFSP2 proteolytic function, implicating the Ufm1 cascade in this form of osteoarthropathy

• Endoplasmic reticulum stress-related disorders:

  • There is increasing evidence that the Ufm1 pathway has a role in the regulation of endoplasmic reticulum (ER) stress responses

  • ER stress is a recognized pathogenic mechanism underlying several forms of osteochondrodysplasia

  • This suggests a potential role for ufsp2 in ER stress-related disorders

• Hematological disorders:

  • Given the importance of ufsp2 in hematopoiesis in zebrafish, there may be implications for hematological disorders

  • UFMylation pathway defects lead to reduced erythrocyte numbers and impaired HSPC survival in zebrafish models

• Premature aging syndromes:

  • The telomere shortening and premature aging phenotypes observed in UFMylation-deficient zebrafish suggest potential implications for human aging-related disorders

  • Further research is needed to explore these connections in human contexts

What are the emerging applications of recombinant ufsp2 in studying cellular stress responses?

Emerging applications of recombinant ufsp2 in studying cellular stress responses include:

• Endoplasmic reticulum stress models:

  • Recombinant ufsp2 can be used to study the link between UFMylation and ER stress responses

  • This is particularly relevant as the Ufm1 pathway appears to be highly expressed in protein-secreting cells and may regulate ER stress responses

• Development of in vitro assay systems:

  • Purified recombinant ufsp2 (wild-type and mutant versions) can be used to develop high-throughput assays for screening compounds that modulate UFMylation

  • Such assays could facilitate the identification of small molecules targeting this pathway for research or therapeutic purposes

• Structure-function studies:

  • Recombinant ufsp2 proteins with specific mutations can help elucidate the structural basis of ufsp2 function

  • This approach has already revealed the importance of the p.Tyr290 residue in the UFSP2 active site

How might advances in understanding ufsp2 function contribute to therapeutic approaches for telomere-related disorders?

Advances in understanding ufsp2 function could contribute to therapeutic approaches for telomere-related disorders in several ways:

• Novel therapeutic targets:

  • The UFMylation pathway represents a previously underexplored mechanism affecting telomere maintenance

  • Components of this pathway could serve as therapeutic targets for conditions characterized by telomere dysfunction

• Biomarkers for telomere-related disorders:

  • Alterations in ufsp2 function or UFMylation of MRE11 could potentially serve as biomarkers for certain telomere-related disorders

  • This might facilitate earlier diagnosis or better monitoring of disease progression

• Mechanism-based interventions:

  • Understanding the specific role of MRE11 UFMylation in recruiting PP1-α and regulating NBS1 phosphorylation provides molecular targets for intervention

  • Strategies aimed at enhancing MRN complex recruitment to telomeres might mitigate the consequences of defective UFMylation

• Combined therapeutic approaches:

  • The synergistic effects observed when UFMylation and telomerase pathways are simultaneously disrupted suggest that combination therapies targeting both pathways might be particularly effective for certain conditions

What control experiments are essential when investigating recombinant ufsp2 activity in vitro?

When investigating recombinant ufsp2 activity in vitro, the following control experiments are essential:

• Enzyme concentration controls:

  • Test increasing concentrations of recombinant ufsp2 to establish dose-dependent activity

  • This approach has been used to demonstrate that wild-type UFSP2 cleaves Ufm1 substrates in a concentration-dependent manner

• Substrate specificity controls:

  • Include non-Ufm1 substrates to confirm the specificity of ufsp2 activity

  • This helps distinguish between specific proteolytic activity and potential non-specific effects

• Mutant enzyme controls:

  • Include catalytically inactive mutants (e.g., ufsp2 with the equivalent of the p.Tyr290His mutation)

  • These serve as negative controls and can also reveal potential dominant-negative effects

• Mixed enzyme assays:

  • Combine wild-type and mutant enzymes in various ratios to assess potential interference or dominant-negative effects

  • This approach revealed that mutant UFSP2 does not interfere with wild-type UFSP2 activity in vitro

• Buffer and reaction condition controls:

  • Include controls for pH, temperature, and cofactor requirements

  • These parameters can significantly affect enzyme activity and should be optimized and controlled

What standardization approaches should be considered when comparing ufsp2 function across different zebrafish studies?

When comparing ufsp2 function across different zebrafish studies, the following standardization approaches should be considered:

• Genetic background standardization:

  • Use well-characterized wild-type strains (e.g., AB, TU, or WIK)

  • Document and report the genetic background of all zebrafish lines used

• Protocol parameter standardization:

  • As demonstrated in toxicity screening studies, protocol parameters can significantly affect experimental outcomes

  • Standardize key parameters such as temperature, feeding regimens, and housing conditions

• Mutant generation and validation:

  • Standardize approaches for generating ufsp2 mutants (e.g., CRISPR-Cas9 targeting strategies)

  • Thoroughly validate mutants at the DNA, RNA, and protein levels to confirm the nature and extent of ufsp2 disruption

• Phenotypic analysis methods:

  • Use consistent methods for assessing key phenotypes (e.g., telomere length, hematopoietic cell counts)

  • When different methods are used (e.g., qPCR vs. qFISH for telomere length), perform validation studies to establish correlation between methods

• Age-matched comparisons:

  • Given the age-dependent nature of many phenotypes associated with ufsp2 dysfunction, ensure comparisons are made between age-matched animals

  • Report precise developmental stages or ages for all analyses