SAE1/SAE2 Human

What is the SAE1/SAE2 complex and what is its primary function?

The SAE1/SAE2 complex is a heterodimeric enzyme that functions as the E1 activating enzyme in the SUMO (Small Ubiquitin-like Modifier) conjugation pathway. SAE1 and SAE2 together form a 113 kDa protein complex that catalyzes the first step in protein sumoylation . This post-translational modification process regulates protein structure and intracellular localization of target proteins .

The complex catalyzes three critical biochemical reactions in SUMO activation:

• Adenylation of the SUMO C-terminus

• Thioester transfer within E1

• Thioester transfer from E1 to E2 conjugating proteins

Structurally, full-length human SAE1 consists of 346 amino acids while SAE2 contains 640 amino acids. SAE2 features three distinct domains: the adenylation domain, the catalytic Cys domain (containing Cys173 responsible for E1-SUMO-thioester bond formation), and a C-terminal ubiquitin-like (UbL) domain that plays a crucial role in E2 recruitment .

How does the SAE1/SAE2 complex interact with different SUMO isoforms?

The SAE1/SAE2 complex interacts with all SUMO isoforms (SUMO-1, SUMO-2, and SUMO-3) but demonstrates different binding affinities. Microscale thermophoresis (MST) analysis has shown that wild-type SAE1/SAE2 has a greater affinity for SUMO1 (Kd = 3.7 ± 1.1 μM) than for SUMO2 (Kd = 14.7 ± 1.8 μM) .

Interaction specificity is mediated through specific residues. For instance, of the 11 SUMO-1 side chains that make direct contact with SAE2, five are strictly conserved across all SUMO isoforms, four are conserved at the amino-acid property level, and two are divergent between SUMO-1 (Asn60 and Arg70) and SUMO-2/3 (Arg and Pro, respectively) .

Interestingly, despite sequence differences, kinetic assays demonstrate that all human SUMO isoforms can be activated by the E1 complex, transferred to an E2-thioester, and conjugated to substrates like RanGAP1 with similar efficiencies in vitro .

What is the structural basis for SAE1/SAE2 function in the SUMO pathway?

The crystal structures of human SAE1/SAE2-Mg·ATP and SAE1/SAE2-SUMO-1-Mg·ATP complexes (resolved at 2.2 and 2.75 Å respectively) provide critical insights into the structural basis of function .

Key structural features include:

• A pseudosymmetric heterodimer formed between the SAE2 adenylation domain (residues 1–158, 384–438) and SAE1 (residues 1–346)

• Three SAE2 domains:

  • Adenylation domain

  • Catalytic Cys domain (residues 159–386) containing Cys173

  • UbL domain (residues 442–549)

• Specific disordered regions not observed in electron density:

  • SAE1 residues 178–203 and 346

  • SAE2 residues 1–3, 219–237, 291–304, and 551–640

In the SAE1/SAE2-SUMO-1-Mg·ATP complex, the SUMO C-terminus remains unmodified within the adenylation site and positioned approximately 35 Å from the catalytic cysteine, suggesting that additional conformational changes are required to facilitate adenylation and thioester transfer .

What are the optimal conditions for expressing and purifying recombinant SAE1/SAE2 complex?

For efficient expression and purification of recombinant SAE1/SAE2, the following methodological approach is recommended:

Expression System:

• Insect cell expression using Sf9 cells has proven effective for producing functional SAE1/SAE2 complex

• Alternatively, bacterial co-expression systems can be used by cloning both human SAE1 and SAE2 genes

Purification Strategy:

• Express the complex with affinity tags (e.g., 10xHis tag) to facilitate purification

• Use proprietary chromatographic techniques for purification

• Formulate in appropriate buffer conditions: 20mM HEPES buffer pH-8.0, 200mM NaCl, and 20% glycerol

Storage Conditions:

• For short-term storage (2-4 weeks): Store at 4°C

• For long-term storage: Store frozen at -20°C

• Avoid multiple freeze-thaw cycles to maintain enzyme activity

The recombinant protein complex typically contains two subunits with SAE1 at 41kDa and SAE2 at 91kDa, which associate to form a functional complex. Purity should exceed 95% as determined by SDS-PAGE analysis .

What assays can be used to measure SAE1/SAE2 enzymatic activity in vitro?

Several complementary assays can be employed to assess SAE1/SAE2 enzymatic activity:

1. E1-Thioester Formation Assay:

• Incubate SAE1/SAE2 with SUMO protein and ATP

• Analyze thioester bond formation by non-reducing SDS-PAGE

• Detect via western blotting with anti-SUMO or anti-SAE2 antibodies

2. ATP-PPi Exchange Assay:

• Measures adenylation activity independent of thioester formation

• Uses [32P]PPi and monitors formation of [32P]ATP

3. E2 Charging Assay:

• Measures transfer of activated SUMO from E1 to E2

• Incubate SAE1/SAE2, SUMO, ATP, and E2 (Ubc9)

• Analyze E2-SUMO thioester formation via non-reducing SDS-PAGE

4. Complete Conjugation Assay:

• Tests the entire pathway from activation to substrate modification

• Includes SAE1/SAE2, SUMO, ATP, Ubc9, and a substrate (e.g., RanGAP1)

• Assesses formation of SUMO-conjugated substrate

For comparative analysis of SUMO isoform preferences, a staged experimental design similar to that shown in Figure 2C of reference can be used, where each step of the pathway is assessed independently using the appropriate assay.

How can I determine SAE1/SAE2-SUMO binding affinities and interaction dynamics?

To determine binding affinities and interaction dynamics between SAE1/SAE2 and SUMO proteins, the following methodological approaches are recommended:

1. Microscale Thermophoresis (MST):

• Label SAE1/SAE2 complex with fluorescent dye

• Titrate with increasing concentrations of unlabeled SUMO protein

• Measure changes in thermophoresis to determine dissociation constants (Kd)

• This method has revealed that wild-type SAE1/SAE2 has higher affinity for SUMO1 (Kd = 3.7 ± 1.1 μM) than SUMO2 (Kd = 14.7 ± 1.8 μM)

2. Surface Plasmon Resonance (SPR):

• Immobilize either SAE1/SAE2 or SUMO on sensor chip

• Flow the binding partner over the surface at various concentrations

• Analyze association and dissociation rates to determine kinetic parameters

3. Isothermal Titration Calorimetry (ITC):

• Directly measures thermodynamic parameters of binding

• Provides enthalpy (ΔH), entropy (ΔS), and binding stoichiometry

4. Mutagenesis Studies:

• Create point mutations at key residues (e.g., SAE2-K164Q)

• Assess effects on SUMO isoform preferences

• This approach has demonstrated that SAE2-K164Q reverses SUMO preference, showing higher affinity for SUMO2 (0.4 ± 0.13 μM) than SUMO1 (28.0 ± 12.19 μM)

What is the role of SAE1/SAE2 in dermatomyositis and how are anti-SAE1/SAE2 autoantibodies detected?

SAE1/SAE2 serves as a diagnostic marker for dermatomyositis (DM) as autoantibodies against these two proteins have been isolated from patients . These autoantibodies represent one of several myositis-specific antibodies that can help in diagnosis and potentially in predicting disease course.

Detection Methods:

• ELISA:

  • Coat plates with purified recombinant SAE1/SAE2 (0.3-0.8 μg/ml)

  • Incubate with patient sera at appropriate dilutions

  • Detect bound autoantibodies with labeled secondary antibodies

  • Perform checkerboard analysis with known positive/negative samples for validation

• Immunodot Assay:

  • Apply purified SAE1/SAE2 to nitrocellulose or PVDF membrane

  • Incubate with patient sera

  • Detect with appropriate secondary antibodies and visualization system

• Western Blot:

  • Separate SAE1/SAE2 by SDS-PAGE

  • Transfer to membrane

  • Probe with patient sera

  • Compare with positive control samples and anti-SAE1/SAE2 antibodies

For clinical applications, recombinant SAE1/SAE2 complex should be of high purity (>95%) and demonstrate proper immunological function by binding to IgG-type human autoantibodies in validation assays .

How does SAE1/SAE2 expression correlate with gastric cancer progression and prognosis?

SAE1/SAE2 expression shows significant correlation with gastric cancer (GC) progression and can serve as a prognostic marker. Key findings include:

Expression Pattern:

• SAE2 mRNA and protein levels are significantly elevated in GC tissues compared to paired non-tumor tissues (P = .011)

• SAE2 protein expression is markedly up-regulated in GC cell lines

• SUMO1-conjugated proteins are also increased in GC tissues

Clinicopathological Correlations:
Higher SAE2 expression in GC significantly correlates with:

• Deeper depth of invasion

• Distant metastasis

• Higher pathological stage (all P < .05)

Methodological Approaches for Analysis:

• Quantigene Plex Assay:

  • For mRNA expression analysis

  • Shows significant correlation with protein expression (P = .017)

• Immunohistochemistry:

  • For protein expression analysis in tissue specimens

  • Scoring system: 0 or 1+ (negative), 2+ and 3+ (positive)

• Statistical Analysis:

  • Chi-squared test for correlations with clinicopathological features

  • Kaplan-Meier method and log-rank test for survival analysis

  • Cox regression for multivariate analysis

How does SAE2 lysine 164 acetylation status regulate SUMO isoform preference?

SAE2 lysine 164 (K164) acetylation status serves as a molecular switch that regulates SUMO isoform preference in the sumoylation pathway. This represents an important regulatory mechanism for controlling which SUMO variants are preferentially conjugated during different cellular processes.

Mechanism of Regulation:

• SAE2-K164 is deacetylated during mitosis in an HDAC6-dependent manner

• The acetylation status of this residue dramatically changes the affinity of SAE1/SAE2 for different SUMO isoforms:

  • Wild-type SAE1/SAE2: Higher affinity for SUMO1 (Kd = 3.7 ± 1.1 μM) than SUMO2 (Kd = 14.7 ± 1.8 μM)

  • SAE1/SAE2-K164Q (acetyl-mimetic): Higher affinity for SUMO2 (Kd = 0.4 ± 0.13 μM) than SUMO1 (Kd = 28.0 ± 12.19 μM)

Molecular Basis:
The crystal structure of SAE1/SAE2-SUMO1 complex reveals proximity between SAE2-K164 and SUMO1-E93. The equivalent residue in SUMO2 is Q89. This difference appears critical for isoform discrimination:

• Swapping these residues (creating SUMO1-E93Q and SUMO2-Q89E) reverses the SUMO preference:

  • SAE1/SAE2-K164Q shifts from SUMO2 bias to SUMO1 bias

  • Wild-type SAE1/SAE2 shifts from SUMO1 bias to SUMO2 bias

This indicates that SAE2-K164 contributes discriminative interactions with the C-terminal regions of SUMO proteins to influence SUMO variant selection during activation and conjugation.

What role does the SAE1/SAE2 complex play in mitotic regulation and chromosome dynamics?

The SAE1/SAE2 complex plays critical roles in mitotic regulation and chromosome dynamics, with implications for genomic stability:

Mitotic Functions:

• SAE2 undergoes deacetylation at K164 during early mitosis to encourage SUMO1 conjugation

• Complementation studies with SAE2-K164Q (acetyl-mimetic) in SAE2-depleted cells reveal:

  • Restricted mitotic SUMO1-conjugates

  • Increased frequency of multipolar spindle formation

Target Proteins:
The Nuclear Mitotic Apparatus protein (NuMA) has been identified as a target for SAE1/SAE2-dependent sumoylation during mitosis . This suggests a mechanism by which the SUMO E1 enzyme regulates mitotic spindle organization and chromosome segregation.

Experimental Approaches:

• Synchronization and Mitotic Analysis:

  • Synchronize cells at specific cell cycle phases

  • Analyze SAE1/SAE2 activity and SUMO conjugation patterns

  • Assess mitotic spindle organization using immunofluorescence microscopy

• SUMO Isoform-Specific Analysis:

  • Use isoform-specific antibodies to distinguish SUMO1 versus SUMO2/3 conjugates

  • Compare wild-type versus SAE2-K164Q effects on sumoylation patterns

  • Correlate with mitotic phenotypes (spindle organization, chromosome segregation)

• Proteomics Approaches:

  • Identify mitosis-specific SUMO targets using mass spectrometry

  • Compare SUMO1 versus SUMO2/3 substrates during mitosis

  • Assess how SAE2-K164 acetylation status affects the mitotic SUMOylome

How can SAE1/SAE2 be targeted therapeutically in cancer and other diseases?

Given the involvement of SAE1/SAE2 in cancer progression and other diseases, therapeutic targeting strategies are being explored:

Rationale for Targeting:

• High SAE2 expression is associated with more aggressive gastric cancer phenotypes and poorer prognosis

• The SUMO pathway regulates many critical cellular processes including DNA damage repair, transcription, and mitosis

• Dysregulated sumoylation contributes to pathogenesis in multiple disease contexts

Potential Therapeutic Approaches:

• Small Molecule Inhibitors:

  • Target the adenylation activity of SAE1/SAE2

  • Inhibit SAE2-SUMO thioester formation

  • Block interaction with E2 enzyme (Ubc9)

• Disruption of Protein-Protein Interactions:

  • Prevent SAE1/SAE2 heterodimer formation

  • Block interactions with SUMO or other pathway components

  • Target the UbL domain of SAE2 that is essential for E2 recruitment

• Targeting Post-Translational Modifications:

  • Modulate SAE2-K164 acetylation status using HDAC inhibitors to alter SUMO isoform preferences

  • This approach could selectively affect specific SUMO-dependent pathways

• Combination Strategies:

  • Sensitize cancer cells to conventional therapies by inhibiting SAE1/SAE2

  • Target synthetic lethal interactions with other pathways

Methodological Considerations:

• High-throughput screening for small molecule inhibitors

• Structure-based drug design guided by crystal structures of SAE1/SAE2

• Validation in cellular and animal models

• Assessment of specificity and potential off-target effects

How do different SUMO isoforms compete for activation by SAE1/SAE2, and what regulates this competition?

The competition between SUMO isoforms for activation by SAE1/SAE2 represents a critical regulatory mechanism in the sumoylation pathway:

Baseline Preferences:

• Wild-type SAE1/SAE2 shows higher affinity for SUMO1 (Kd = 3.7 ± 1.1 μM) than SUMO2 (Kd = 14.7 ± 1.8 μM)

• Despite these differences in affinity, all SUMO isoforms (SUMO-1, -2, and -3) can be activated, transferred to E2, and conjugated to substrates with similar kinetics in vitro

Regulatory Mechanisms:

• Post-translational Modifications of SAE1/SAE2:

  • SAE2-K164 acetylation status dramatically shifts SUMO isoform preference

  • Deacetylation during mitosis promotes SUMO1 conjugation

• Structural Determinants:

  • Specific residues in the C-terminal regions of SUMO proteins influence recognition

  • Key residue pairs include SAE2-K164 interacting with SUMO1-E93/SUMO2-Q89

• Concentration-Dependent Competition:

  • Relative abundance of SUMO isoforms in specific cellular compartments

  • Local concentrations may override intrinsic affinity differences

• Cell Cycle Regulation:

  • Temporal regulation of SAE1/SAE2 modifications during cell cycle progression

  • HDAC6-dependent deacetylation of SAE2-K164 in early mitosis

Experimental Approaches to Study Competition:

• In Vitro Competition Assays:

  • Mix different ratios of SUMO isoforms with limiting SAE1/SAE2

  • Measure activation rates for each isoform

  • Assess how modifications of SAE1/SAE2 affect competition outcomes

• Cell-Based Competition Systems:

  • Express tagged versions of different SUMO isoforms

  • Monitor their conjugation under various conditions

  • Evaluate how modulating SAE1/SAE2 (e.g., with K164 mutations) affects the balance

• Mathematical Modeling:

  • Develop kinetic models of SUMO isoform competition

  • Incorporate parameters for concentrations, affinities, and enzymatic rates

  • Predict how perturbations would affect the system

What new technologies are being developed to study SAE1/SAE2 function and dynamics in living cells?

Advanced technologies are enabling unprecedented insights into SAE1/SAE2 function and dynamics:

CRISPR-Based Approaches:

• CRISPR/Cas9 gene editing to introduce endogenous tags or specific mutations (like SAE2-K164Q)

• CRISPR interference (CRISPRi) or activation (CRISPRa) for temporal control of expression

• Base editing for precise modification of specific residues without double-strand breaks

Live Cell Imaging:

• FRET-based sensors to monitor SAE1/SAE2-SUMO interactions in real-time

• Photoactivatable or photoswitchable fluorescent proteins to track protein dynamics

• Single-molecule tracking to analyze diffusion, binding kinetics, and complex formation

Proximity Labeling:

• BioID or TurboID fusions to SAE1/SAE2 components to identify transient interactors

• Spatial-specific variants to examine compartment-specific interactions

• Temporal control using optogenetic or chemical biology approaches

Structural Biology Advances:

• Cryo-electron microscopy to visualize conformational changes during the catalytic cycle

• Hydrogen-deuterium exchange mass spectrometry to map dynamic protein interfaces

• Integrative structural biology combining multiple data types for complete models

These technologies will help address outstanding questions about how SAE1/SAE2 function is regulated in space and time within living cells during normal physiology and disease states.

How does the interplay between SAE1/SAE2 and other post-translational modification pathways create regulatory networks?

The SAE1/SAE2 complex does not function in isolation but participates in complex regulatory networks with other post-translational modification (PTM) pathways:

Cross-Regulation with Acetylation:

• SAE2-K164 acetylation status regulates SUMO isoform preference

• HDAC6-dependent deacetylation during mitosis promotes SUMO1 conjugation

Interplay with Ubiquitination:

• SUMO-targeted ubiquitin ligases (STUbLs) recognize SUMOylated proteins

• Mixed SUMO-ubiquitin chains create complex signaling platforms

• Competition between SUMO and ubiquitin for the same lysine residues

Phosphorylation Networks:

• Potential phosphorylation of SAE1/SAE2 components by cell cycle kinases

• Phosphorylation of SUMO substrates can enhance or inhibit SUMOylation

• Phosphorylation-dependent SUMO-interaction motifs (SIMs) in target proteins

Methodological Approaches:

• Multi-PTM Proteomics:

  • Enrichment strategies to isolate proteins with multiple modifications

  • Mass spectrometry to identify co-occurring PTMs

  • Bioinformatic analysis of PTM crosstalk networks

• Targeted Mutation Analysis:

  • Mutate specific modification sites (e.g., SAE2-K164) and analyze effects on other PTMs

  • Create modification-deficient variants of SUMO substrates

  • Assess functional consequences in relevant biological contexts

• Systems Biology Approaches:

  • Network analysis of PTM crosstalk

  • Mathematical modeling of dynamic PTM interactions

  • Prediction and validation of emergent properties from PTM networks

Understanding these regulatory networks will provide insights into how cells integrate multiple signaling inputs and coordinate complex processes like cell division, stress responses, and differentiation.