Linear Ubiquitination

New Approach to Discovering Physiological Roles and Disease Relevance

  • Fig. 1: Schematic representation of (de)ubiquitination system.Fig. 1: Schematic representation of (de)ubiquitination system.
  • Fig. 1: Schematic representation of (de)ubiquitination system.
  • Fig. 2: Various approaches to detect ubiquitination in vivo.
  • Fig. 3: Overview of INT-Ub.7KR approach.
Protein modifications by a small protein ubiquitin control diverse cellular processes ranging from protein degradation to cell survival. Modification by a specific type of ubiquitin chains (linear ubiquitination) regulates key cellular processes such as inflammatory response, survival and autophagy. Till date, several methods for identification of ubiquitinated proteins have been successfully developed and applied, but none of them is suitable for the discovery of linear ubiquitin-modified substrates. Not surprisingly, only a few proteins modified by linear ubiquitin chains are known, including the key regulator of inflammation NEMO. We have recently developed and applied a novel approach for the enrichment of linear ubiquitination targets, by combining mass spectrometry and lysine-less internally tagged ubiquitin (INT-Ub.7KR) variant, which allowed us the identification of novel substrates of linear ubiquitination. Here, we discuss the current approaches and future directions in analysing linear ubiquitination networks to further decipher their physiological and disease relevance.
Post-translational modifications of proteins regulate protein functions in numerous ways. One such dynamic and reversible modification is ubiquitination, which regulates protein stability, cell cycle, DNA transcription and repair, cell death and immune response. Ubiquitin can be attached to other proteins through the coordinated activity of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes (fig. 1), and the process can be reversed through the activity of more than 100 proteases known as deubiquitinating enzymes (DUBs) (fig. 1), which remove ubiquitin from its target [1]. 
Ubiquitination is a covalent modification of target proteins, which relies on the formation of an isopeptide bond between the α-carboxyl group of the terminal glycine (Gly) of ubiquitin and the ε-amino group of lysine (Lys) residue of the substrate. Modification by a single ubiquitin moiety (monoubiquitination) is very common and regulates important cellular processes, such as DNA repair and receptor endocytosis.

Since ubiquitin itself contains seven Lys residues (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48 and Lys63), it can itself be ubiquitinates at seven different positions, forming ubiquitin chains of different architectures and functions in the cell [2]. 

In 2006, additional type of ubiquitin chains was discovered, in which N-terminal methionine (Met) of one ubiquitin molecule forms a peptide bond with the C-terminal Gly residue of another ubiquitin, generating head-to-tail, or linear ubiquitin chains [3]. 
Distinct features of various ubiquitin chains are recognized by proteins containing ubiquitin-binding domains (UBDs) [4], which act as “readers” of various ubiquitin modifications (fig. 1), and help determine exact cellular consequences of specific ubiquitination events. 
Linear Ubiquitination Has Important Physiological Roles, but Not Many Known Substrates
Among all the known ubiquitin modifications, linear ubiquitination is one of the least studied. There are more than 600 E3 ligases in human genome, yet only one E3 ligase complex was shown to generate linear ubiquitin chains. LUBAC (linear ubiquitin assembly complex) consists of three proteins: catalytic subunit RNF31 (HOIP) and adaptor proteins RBCK1 (HOIL-1L) and SHARPIN [5]. Several DUBs can cleave linear ubiquitin: CYLD, USP10 and OTULIN, but only the latter exhibits exclusive specificity for linear linkages [6].
Although levels of linear ubiquitination are almost undetectable in resting cells, specific stimuli (such as cytokines TNFα and IL-1β, or bacterial infection) efficiently induce LUBAC-dependent linear ubiquitination in vivo [7-9]. Historically, the first and the most studied signalling pathway regulated by linear ubiquitination is the canonical NFκB pathway, which plays a key role in regulating immunity, inflammatory response, cell differentiation and survival. Linear ubiquitination of NEMO, which acts as the regulatory subunit of the IKK (IκB kinase) complex, is essential for the activation of the pathway [10].
Till date, linear ubiquitination machinery and its targets have been shown to regulate the adaptive and innate immunity, autoinflammation, lymphocyte development, genotoxic stress response, autophagy and Parkinson´s disease [11]. Unfortunately, the molecular mechanisms and exact substrates of linear ubiquitination remain largely unknown. 
Standard Approaches Are Not Optimal for the Identification of Linear Ubiquitin Targets In Vivo 
Over the last decades, multiple approaches have been developed to detect ubiquitination in the cell. The use of N-terminally tagged ubiquitin variants enables enrichment and subsequent identification of mono- and Lys-linked ubiquitination substrates (fig. 2A). Nevertheless, this approach is not suitable for linear ubiquitination, due to the lack of free N-terminal Met required for the formation of linear ubiquitin chain.
Tandem ubiquitin-binding entities (TUBEs) are often used as enrichment tools for ubiquitinated proteins [12] with specificity being achieved by the use of ubiquitin linkage-specific UBD tandems (fig. 2B). Due to mild lysing conditions necessary to ensure TUBE-ubiquitin interactions (interactions between ubiquitin and UBDs are usually in micromolar range), such approach combined with mass spectrometry generates a long list of proteins, which consists of both ubiquitinated proteins and their non-covalent interaction partners, making the actual validation of modified proteins very laborious and complex. Therefore, although linear ubiquitin-specific TUBEs exist, their applicability is mainly limited to the validation of putative linear ubiquitin-modified proteins.
Immunoprecipitation approach known as ubiquitin remnant immunoaffinity profiling (fig. 2C) is a widely used method for the enrichment of total pools of ubiquitinated proteins (13). It is based on the use of specific antibodies recognizing the remnants of linkers between ubiquitin and target protein obtained after sample trypsin digestion (Gly-Gly-Lys), followed by mass spectrometry analysis. Such approach enables simultaneous detection of thousands of ubiquitination sites, facilitating the study of proteome-wide ubiquitination [14-15]. Since these antibodies cannot recognize linear ubiquitination signature peptide Gly-Gly-Met-Gln-Ile-Phe-Val-Lys (obtained after trypsin digestion of linear ubiquitin-modified substrates), such approach cannot be used for the identification of linear ubiquitin-modified targets.
Most of the antibodies raised against ubiquitin can recognize single ubiquitin molecules or ubiquitin chains and cannot distinguish between various ubiquitin linkages. In general, there are not many ubiquitin chain-specific antibodies and they often preferentially (but not exclusively) recognize certain ubiquitin chains under well-defined experimental conditions [16,17]. 
Internally Tagged Lysine-Less Ubiquitin as Tool for Detection of Linear Ubiquitin-Modified Proteins
We have recently extended the collection of existing methods for studying ubiquitinated proteins by the approach that was specifically developed for the identification of linear ubiquitin-modified substrates (fig. 3) [7].
Insertion of the small STREP-tag within ubiquitin molecule generated internally tagged ubiquitin (INT-Ub) (fig. 3A). Having free N-terminus, such ubiquitin variant can be incorporated into any ubiquitin chain and due to presence of STREP-tag, it can be pulled-down (PD) under highly denaturing conditions from complex cellular lysates. Importantly, presence of internal tag does not affect neither structural nor functional features of ubiquitin, as INT-Ub undergoes all ubiquitin-specific processes: incorporation into ubiquitin chains, interaction with various UBDs, as well as recognition and cleavage by DUBs (fig. 3B).
Further modification of INT-Ub by substitution of all the Lys residues with Arg resulted in the generation of INT-Ub.7KR variant, which can be assembled only into linear ubiquitin chains. Applicability of INT-Ub.7KR was confirmed by the identification of linear ubiquitin-modified NEMO in INT-Ub.7KR PD upon TNFα stimulation. 
By combining mass spectrometry with INT-Ub.7KR, we identified numerous novel linear ubiquitin-modified substrates in TNFα-stimulated cells. Extensive validation confirmed that several selected MS candidates (i.e. PLAA, SEPT2, BRAP and TRAF6) are indeed linearly ubiquitinated [7].  Interestingly, newly established INT-Ub.7KR approach extended current knowledge on the well-studied NFκB regulator TRAF6, by identifying LUBAC as the first E3 ligase complex capable of ubiquitinating TRAF6, and by discovering that TRAF6 linear ubiquitination is essential for the proper activation of NFκB pathway.
Understanding intricate regulation of cellular processes by various post-translational modifications, with emphasis on their crosstalk and specific roles in development and disease progression, is of utmost importance. Development of reliable, sensitive and robust tools significantly improves our understanding of such processes. By using our newly developed approach [7], we have identified several novel linear ubiquitin-modified proteins, significantly expanding the number of known linear ubiquitin targets, which can be now further studied by specialized laboratories. Moreover, our approach theoretically enables identification of linear ubiquitin targets generated by still undiscovered linear ubiquitin-specific E3 ligases, as well as identification of linear ubiquitin targets modified under specific cellular conditions. 
Katarzyna Kliza1 and Koraljka Husnjak1
1 Goethe University Frankfurt , Medical Faculty, Institute of Biochemistry II, Frankfurt am Main, Germany
Goethe University Frankfurt
Medical Faculty
Institute of Biochemistry II
Ubiquitin Signalling Group
Frankfurt am Main, Germany

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