Cellular ubiquitin pool dynamics and homeostasis

Abstract

Ubiquitin (Ub) is a versatile signaling molecule that plays important roles in a variety of cellular processes. Cellular Ub pools, which are composed of free Ub and Ub conjugates, are in dynamic equilibrium inside cells. In particular, increasing evidence suggests that Ub homeostasis, or the maintenance of free Ub above certain threshold levels, is important for cellular function and survival under normal or stress conditions. Accurate determination of various Ub species, including levels of free Ub and specific Ub chain linkages, have become possible in biological specimens as a result of the introduction of the proteomic approach using mass spectrometry. This technology has facilitated research on dynamic properties of cellular Ub pools and has provided tools for in-depth investigation of Ub homeostasis. In this review, we have also discussed the consequences of the disruption of Ub pool dynamics and homeostasis via deletion of polyubiquitin genes or mutations of deubiquitinating enzymes. The common consequence was a reduced availability of free Ub and a significant impact on the function and viability of cells. These observations further indicate that the levels of free Ub are important determinants for cellular protection.

Fig. 1.. Dynamic properties of cellular Ub pools. Conjugation of protein substrates (S) with Ub by a series of enzymes (E1-E3) and deconjugation by DUBs are in dynamic equilibrium. Ub conjugation results in monoubiquitination or polyubiquitination with different types of chain linkages. Ub conjugates or polyubiquitinated protein substrates with specific chain linkages (e.g., K48-linked chains) are targeted to the 26S proteasome for degradation. During degradation of substrates, Ub chains are removed en bloc by a specific DUB (e.g., POH1) and are further processed to free Ub. The free Ub pool is maintained by de novo Ub synthesis via expression of four different ubiquitin genes and by recycling or removal of Ub from Ub conjugates via actions of various DUBs (e.g., UCH37, USP14). Under certain conditions, Ub itself can also be degraded by the proteasome. See the text for details.

Fig. 2.. Mass spectrometry-based approach to quantify cellular Ub pools. (Left) The amount of protein substrates (S) with specific Ub chains can be determined accurately via AQUA. After enriching ubiquitinated substrates, branched signature peptide fragments are generated by trypsin digestion since trypsin cannot cleave modified Lys. A known amount of stable isotope-labeled synthetic peptide standard (AQUA standard) is added into samples during trypsin digestion. Then, by comparing ion intensities, the signature peptide fragments are quantified after LC-MS analysis. For simplicity, only the K48-linked chains are shown. (Right) The amount of free Ub, total Ub, and protein substrates (S) with Ub chains can be quantified via Ub-PSAQ. Samples are mixed with a known amount of stable isotope-labeled protein recovery standard. Half of the sample is affinity-captured with a BUZ domain that specifically binds free Ub, followed by a UBA domain that specifically binds polyUb chains. The other half of the sample is deubiquitinated by treatment with Usp2-cc and is affinity-captured with the BUZ domain to quantify the total amount of Ub. After elution, the AQUA standard is added during trypsin digestion, and LC-MS analysis is carried out. Ion intensities of endogenous peptide fragments are compared to both the AQUA standard (for quantification of peptide fragments in the eluate) and protein standard (for determination of recovery from the affinity column), and the peptide fragments are quantified.

Fig. 3.. Various types of ubiquitination and the specific role of Ub chain linkages. Ub is composed of 76 amino acids and forms isopeptide linkages between the carboxyl group of Gly76 residue and ε-amino group of Lys residue in substrate or in another Ub molecule. The substrate can be monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated with different types of chain linkages. The 1^st Met and seven Lys residues in Ub (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63) can all form specific chain linkages with distinct conformations and can exert specific (or unknown) functions. Although it is rare, heterogeneous and branched/forked chain linkages are also possible.