Biology subjects

Lanz, A.

Publications and source records attributed to Lanz, A..

3 recordsLinked to original sources

bioRxiv2022

SUMO4 regulates DNA double-strand break repair independently of conjugation.

The amplitudes of small-modifier protein signalling through ubiquitin and the Small Ubiquitin-like Modifiers, SUMO1-3, are critical to the correct phasing of DNA repair protein accumulation, activity, and clearance, and for the completion of mammalian DNA double-strand break (DSB) repair. However, how SUMO-conjugate signalling in the response is delineated is poorly understood. At the same time, the role of the non-conjugated SUMO protein, SUMO4, has remained enigmatic. Here we reveal that SUMO4 is required to prevent excessive DNA-damage-induced SUMOylation and deleterious over-accumulation of RAP80. Mechanistically we show SUMO4 acts independently of its conjugation and potentiates SENP1 catalytic activity. These data identify SUMO4 as a SUMO deconjugation component and show SUMO4:SENP1 are critical regulators of DNA-damage-induced SUMO signalling. HighlightsO_LIThe roles of the SUMO family members, SUMO1-4, in DSB repair are not redundant. C_LIO_LISUMO4 conjugation is deleterious. C_LIO_LISUMO4 promotes SENP1 catalytic activity. C_LIO_LISUMO4:SENP1 restrict SUMO-signalling and accumulation of the BRCA1-A complex. C_LI

molecular biology↗

bioRxiv2022

SUMO monoclonal antibodies vary in sensitivity, specificity, and ability to detect SUMO conjugate types.

Monoclonal antibodies (MAb) to members of the Small Ubiquitin-like modifier (SUMO) family are essential tools in the study of cellular SUMOylation. However, many reagents are poorly validated, and the choice of which antibody to use for which detection format is without an evidence base. Here we test twenty-four anti-SUMO monoclonal antibodies for sensitivity and specificity to SUMO1-4 in monomeric and polymeric states in dot-blots, immunoblots, immunofluorescence and immunoprecipitation. We find substantial variability between SUMO MAbs for different conjugation states, detection of increased SUMOylation in response to thirteen different stress agents and as enrichment reagents for analysis of SUMOylated RanGAP1 or KAP1. All four anti-SUMO4 monoclonal antibodies tested cross-reacted with SUMO2/3, and several SUMO2/3 monoclonal antibodies cross-reacted with SUMO4. ~10% of tested monoclonal antibodies produced specific results across multiple applications.

molecular biology↗

bioRxiv2022

Modification of the SUMO activating enzyme subunit SAE2 directs SUMO isoform bias required for mitotic fidelity.

Mammalian cells possess three conjugatable SUMO variants: SUMO1 and the largely indistinguishable SUMO2 and SUMO3 (designated SUMO2/3). Some SUMOylated substrates are modified by both SUMO1 and SUMO2/3, while others show biased modifications towards SUMO1 or SUMO2/3. How preferential SUMO protein conjugation is coordinated is poorly understood. Here, we examine a modification of the catalytic component of the human SUMO Activation Enzyme, SAE2. We observe that lysine 164 of SAE2 is deacetylated during mitosis in an HDAC6-dependent manner. We find that an acetyl-analogue mutant, SAE2-K164Q, biases the activation and conjugation of SUMO2>SUMO1 and discriminates SUMO1 and SUMO2/3 through their C-terminal tails. Complementation of SAE2-depleted or inhibited cells with SAE2-K164Q restricts mitotic SUMO1-conjugates and increases multipolar spindle formation. We confirm the SUMO E1-dependent modification of the nuclear mitotic apparatus, NuMA, and find that the mitotic defects of both SAE2-K164Q complemented cells and HDAC6-inhibitor-treated cells are corrected by either over-expression of SUMO1 or by expression of a GFP-SUMO1-NuMA-K1766R fusion protein. Our observations suggest a model in which the SAE1:SAE2 enzyme is deacetylated on early mitosis to encourage the conjugation of SUMO1 to support mitotic fidelity. These surprising data reveal that the SUMO-activating enzyme can bias SUMO variant conjugation.

molecular biology↗