Antibodies to Ubiquitin-like Proteins

Introduction: Ubiquitin-like proteins fall into two classes: Ubiquitin-like modifiers (UBLs) and Ubiquitin-domain proteins (UDPs).

The first class, ubiquitin-like modifiers, function as modifiers in a manner analogous to that of Ubiquitin, which influences protein turnover in a cell by closely regulating the degradation of specific proteins (please see “Focus On Ubiquitin”). Examples of UBLs are SUMO, Rub1 (also called Nedd8), Apg8 and Apg12.

Proteins of the second class include parkin, RAD23 and DSK2, are designated ubiquitin-domain proteins. These proteins contain domains that are related to ubiquitin but are otherwise unrelated to each other. In contrast to UBLs, UDPs are not conjugated to other proteins.

Apg12 is synthesized in its mature form and seems to have one target, Apg5. Almost all Apg12 molecules are conjugated with Apg5. In yeast, autophagy, the delivery of cytoplasmic components to the lysosome/vacuole for degradation, requires a ubiquitin-like protein conjugation system, in which Apg12 is covalently bound to Apg12-Apg5 and Apg16.

Apg8 exists in two forms, one is membrane bound through a phospholipid. Lipidation/ activation of Apg8 is mediated by Apg7 and transferred to Apg3 and finally forms a conjugate with phosphatidyl-ethanolamine (PE). Apg4 cleaves Apg8–PE, releasing Apg8 from membrane. Morphological studies show that Apg8 localizes on the membrane of intermediate structures of the autophagosome; this transient association seems to be essential for formation of the autophagosome.

Hub1 ("Homologous to Ubiquitin") may function as a modifier (see fig. 1) but its role is unclear because it lacks the double glycine motif characteristic for ubiquitin and ubiquitin-like modifiers. Recently cell polarity factors Sph1 and Hbt1 have been identified as in vivo targets of Hub1 conjugation. HUB1 has close homologs in other species including humans. The human homologs is called Ubl5.

Rub1 and the corresponding human homolog Nedd8 are activated by the E1 ubiquitin-activating enzyme UBA2, that forms isopeptide linkages between thio esters. Rub1 modification may regulate SCF function or localization.

Urm1 is a newly identified ubiquitin related modifier. Urm 1 is a 99-amino acid protein terminated with glycine-glycine. Target proteins are conjugated to Urm1 via its C-terminal glycine. Initially Urm1 forms a thioester with a novel E1-like protein, Uba4

ISG15 (Interferon Stimulating Gene-15) also known also as UCRP (ubiquitin cross-reactive protein) shows no amino acid sequence homology to cytokines and is synthesized as a precursor that is activated through processing by a thiol protease. ISG15 is secreted by monocytes and lymphocytes. ISG15 has been shown to modulate immune cell function. It possesses activities of cytokines and induces production of IFN-gamma. It enhances proliferation and functions of natural killer and LAK cells.

Covalent modification of cellular proteins by the ubiquitin-like modifier SUMO (small ubiquitin-like modifier) regulates various cellular processes, such as nuclear transport, signal transduction, stress responses and cell cycle progression. But, in contrast to ubiquination, sumoylation does not tag proteins for degradation by the 26S proteasome, but rather seems to enhance stability or modulate their subcellular compartmentalization.

Fig.1: Conjugation pathways for ubiquitin and UBLs. Most modifiers mature by proteolytic processing from inactive precursors (a; amino acid). Arrowheads point to the cleavage sites. Ubiquitin is expressed either as polyubiquitin or as a fusion with ribosomal proteins. Conjugation requires activating (E1) and conjugating (E2) enzymes that form thiolesters (S) with the modifiers. Modification of cullins by RUB involves SCF(SKP1/cullin-1/F-box protein) /CBC(cullin-2/elongin B/elonginC) -like E3 enzymes that are also involved in ubiquitination. In contrast to ubiquitin, the UBLs do not seem to form multi-UBL chains. UCRP(ISG15) resembles two ubiquitin moieties linked head-to-tail. Whether HUB1 functions as a modifier is currently unclear. APG12 and URM1 are distinct from the other modifiers because they are unrelated in sequence to ubiquitin.

Antibody Tools for the Detection of Ubiquitin-like proteins

Acris Antibodies offers a wide range of antibodies for the detection of proteins belonging to the Ubiquitin-like modifier family. These antibodies are available in various formats and can be used in immunological methods like Western blotting or ELISA.

Fig.2: Immunoblot of Hub1 fusion protein. Anti-Hub1 antibody generated by immunization with recombinant yeast Hub1 was tested by immunoblot against yeast lysates expressing the Hub1-GFP fusion protein and other UBL fusion proteins. All UBLs possess limited homology to Ubiquitin and to each other, therefore it is important to know the degree of reactivity of each antibody against each UBL. Panel A shows total protein staining using ponceau. Panel B shows positions of free GFP or GFP containing recombinant proteins present in each lysate preparation after reaction with a 1:1,000 dilution of Acris anti-GFP (code #R1091P) followed by reaction with a 1:15,000 dilution of HRP Donkey-a-Goat IgG (code # R1254HRP). Panel C shows specific reaction with Hub1 using a 1:500 dilution of Acris IgG fraction of Rabbit-anti-Hub1 (Yeast) followed by reaction with a 1:15,000 dilution of HRP Goat-a-Rabbit IgG (code # R1454HRP). All primary antibodies were diluted in TTBS buffer supplemented with 5% non-fat milk and incubated with the membranes overnight at 4°C. Yeast lysate proteins were separated by SDS-PAGE using a 15% gel. This data indicates that anti-Hub1 is highly specific and does not cross react with other UBLs

Available Antibody Panel to UBLs

APG4B antibody, APG5L, APG7, APG8, APG12, Hub1, Rub1, URM1 (Urm1), ISG15, SUMO (Sumo), SUMO-1 (Sumo1), SUMO-2/3 (Sumo2/3), SUMO-3 (Sumo3) antibody.