Tools to Examine Ubiquitination of Proteins

Abstract

It’s no secret that proteins frequently are modified post translation. Most such changes involve small, simple chemical groups: phosphorylation, methylation, acetylation and the like. Glycosylation, the addition of intricate and sometimes branched polysaccharides to a protein chain, is one exception; ubiquitination is another.

Ubiquitin is not so much a modification, per se, as it is another protein: a 76-amino acid tag whose conjugation to substrate proteins (via substrate lysines) often, though not always, marks them for destruction by proteasomes—a complex of proteases that carries out the degradation. In some cases, target proteins are tagged with single ubiquitin molecules; in other cases, one ubiquitin couples to another in long polyubiquitin chains. But in any event, the effect can be profound.

“Ubiquitination has emerged as a central regulatory mechanism that controls not only protein stability, but also localization, interactions and functional activity for a vast number of protein substrates,” wrote the authors of one 2005 review [1].

Among ubiquitin’s targets are IkB (the inhibitory protein that prevents the translocation of the transcription factor NF-kB into the nucleus) and various components of the mitochondrial outer membrane, which are “painted” by a ubiquitin ligase called PARKIN to mark damaged organelles for destruction, according to Bradley Brasher, general manager at Boston Biochem, a division of Bio-Techne that bills itself as “the world’s leading producer of ubiquitin-related research products.” Dysregulation of PARKIN activity causes an accumulation of damaged mitochondria and is associated with Parkinson’s disease.

Today, researchers have a variety of commercial tools and protocols to aid in such studies. Here are some of your options.

Enzymes and inhibitors 

Ubiquitination is the result of a three-enzyme cascade. E1 activators covalently bind to free ubiquitin via a thioester linkage and transfer that protein to E2 conjugating proteins. The E3 ubiquitin ligase then facilitates the transfer of ubiquitin from E2 to its substrate protein. Each enzyme in this pathway exists in multiple forms in the human genome, Brasher notes—a handful of E1s, 35 or 40 E2s and as many as 600 E3s.

On the other side of the equation, deubiquitinating enzymes (DUBs)—of which the human genome encodes some 90 to 100 variants—remove ubiquitin chains from their protein targets.

Reagent vendors now offer a broad palette of tools for manipulating and studying these enzymes both in vitro and in vivo.

Recombinant enzymes are available from several companies, including Boston Biochem. Researchers can use those reagents to drive drug-screening applications or to determine which specific enzyme ubiquitinates (or deubiquitinates) their protein of interest. “That can be tricky to answer [in vivo],” Brasher says. “So they can test it in the test tube.”

That’s not to say those questions are easy to answer in vitro. As when trying to match a phosphorylation event to a specific kinase or phosphatase, it is far easier to determine that a protein has been ubiquitinated (for instance, via Western blotting with an anti-ubiquitin antibody) than to determine which E3 ligase places that post-translational modification, or which DUB removes it, Brasher notes.

Vendors also sell ubiquitin itself, both as a monomer and in a variety of polyubiquitinated forms. These reagents can be used, for instance, to assess DUB specificity. UbiQ sells a panel of eight di-ubiquitin conjugates for use in enzyme assays and protein-protein interaction studies. Boston Biochem sells di-, tri- and tetra-ubiquitin chains (and sometimes longer) linked through six of the protein’s seven lysine residues: K6, K11, K29, K33, K48 and K63. K48 polyubiquitin chains are typically associated with proteasome degradation, according to company literature, and K63 chains “[have] been implicated in several non-degradative processes such as receptor endocytosis and sorting, translation, DNA damage repair, the stress response and signaling in the NFκB pathway.” But for the other linkages, “the biology isn’t always understood,” Brasher says.

Enzyme assay kits also are available, as are specialty substrates such as quenched fluorophore-conjugated forms of ubiquitin. Available from UbiQ and Boston Biochem, among others, these latter reagents enable researchers to assess DUB activity in microtiter plates, for instance in drug-screening studies.

And then there are the inhibitors of these various enzymes, including the proteasome itself, which block degradation of ubiquitinated proteins. According to Chandra Mohan, senior manager for technical content and training at MilliporeSigma, two recently approved pharmaceuticals for the treatment of multiple myeloma—Kyprolis from Amgen and Ninlaro from Takeda—both work by inhibiting the mammalian proteasome. But on the research front, he says, the most popular proteasome inhibitors currently in use are MG-132 and lactacystin, which can be used to determine, for instance, what happens if researchers block the turnover of a specific transcription factor.

Antibodies

Wide arrays of antibodies for ubiquitin research are available, including those reactive toward the ubiquitin monomer and some polyubiquitin structures. But one antibody in particular, which targets the ubiquitin-substrate linkage itself, is particularly useful for proteomic studies.

In typical bottom-up analyses, crude protein extracts are digested into peptides with trypsin. But as ubiquitin is also a protein, trypsin digestion also chews away the ubiquitin tag. As a result, peptides to which ubiquitin was conjugated are marked with a three-amino acid tag: a diglycine moiety (from ubiquitin) hanging off the conjugation point lysine, a motif called K-e-GG.

With these antibodies, researchers can enrich ubiquitinated peptides sufficiently to detect and identify them in the mass spectrometer, thus deciphering what might be called the “ubiquitinome.”

Several companies offer antibodies that target that motif, including Cell Signaling Technologies, MilliporeSigma and Lucerna. With these antibodies, researchers can enrich ubiquitinated peptides sufficiently to detect and identify them in the mass spectrometer, thus deciphering what might be called the “ubiquitinome."

Sean Beausoleil, associate director of discovery proteomics at Bluefin Biomedicine, a spin-off of Cell Signaling Technologies, uses CST’s K-e-GG-specific antibody to advance his company’s cancer drug-discovery efforts and analyzes the resulting peptides on a Thermo Fisher Scientific Orbitrap Fusion Lumos mass spectrometer.

Lumos, Beausoleil explains, offers two key advantages. The first is sensitivity. Featuring a brighter ion source and more efficient mass filter, the Lumos—Thermo’s newest, top-of-the-line Orbitrap—is about five times more sensitive than its predecessor, the Orbitrap Fusion, says Graeme McAlister, senior scientist for life sciences mass spectrometry at Thermo Fisher Scientific.

That’s particularly important for ubiquitin research, McAlister explains, because ubiquitinated peptides are relatively rare, even following antibody enrichment: “The reason [ubiquitin mass spectrometry] is tricky is because these proteins are often flagged for degradation, so there’s not a lot of them around.”

The other advantage, according to Beausoleil, is the instrument’s compatibility with sample multiplexing, for instance using Thermo’s TMT 10-plex isobaric tags, which allows him to combine multiple patient samples per run and thereby identify subtle similarities and differences. Lumos, he concludes, “has been a game-changer for us.”

Researchers studying protein ubiquitination have available to them a selection of both molecular and biochemical assays—plus the ability to add mass spectrometry analysis to gain a better understanding of how and why proteins become ubiquitinated. Ultimately, this will lead to a better understanding of how the cell controls and regulates its translational events, especially during normal and disease or stressed growth states.

 

Reference:

[1] Kirkpatrick, DS, Denison, C, Gygi, SP, “Weighing in on ubiquitin: the expanding role of mass-spectrometry-based proteomics,” Nat Cell Biol, 7:750-7, 2005. [PMID:16056266