300364 VO Research Topics in Molecular Microbiology, Microbial Ecology and Immunobiology

Prof. Gijs Versteeg – Ubiquitin and Ubiquitin-like molecules

Innate immunity – angeborene Immunität (rapid response)Adaptive immunity – erworbene Immunität (slow response)

Innate immunity is essential for the survival of the organisms that were infected. It is activated rapidly and it makes sure that you survive long enough before the adaptive immunity is activated and virus cleared. Knockout mouse without innate immunity cannot survive, whereas mice without adaptive immunity survive normally, just have a bit prolonged infection.Innate immunity recognizes unprocessed microbial molecules. The principal components of the innate immunity are: physical and chemical barriers (e.g. epithelia and chemicals produced at epithelial surface), phagocytic cells (neutrophils, macrophages), dendritic cells (they are essential, because they later present antigens and present if to T and B cells, and eventually activate adaptive immune response, and are considered professional phagocytes together with macrophages and neutrophils, DCs, in contrast, have developed means to 'preserve' useful information from the ingested particles that serve to initiate adaptive immune responses; both phagosomal degradation and acidification are much lower in DCs than in macrophages or neutrophils. Reduced degradation results in the conservation of antigenic peptides and in their increased presentation on major histocompatibility complex class I and II molecules) and NK cells; blood proteins including complement system and other mediators of inflammation; proteins called cytokines. Innate immunity cannot distinguish between different strains, but can distinguish between groups of pathogens (families). Innate immunity recognizes so called PAMPs (pathogen-associated molecular patterns). They can recognize these PAMPs either inside of the cell (e.g. cytosol) or outside of the cell (e.g. cell wall). PAMPs are molecules that are unique to groups of related microorganisms. There are many types of PAMPs: peptidoglycan and lipoteichoic acid in Gram+ bacteria, lipopolysaccharide in Gram- bacteria, 5’-ppp-RNA, dsRNA and other patterns associated with viruses. Innate immunity can also recognize DAMPs (damage-associated molecular patterns), that may be produced as a result of cell damage caused by infection or by any of myriad reasons. Innate immunity can distinguish between self and non-self, but does not build up any memory whatsoever. PAMPs and DAMPs are recognized by already mentioned cells of innate immunity, which possess different cellular receptors for PAMPs (and DAMPs) that are called PRR –pattern recognition receptors.

TOLL-like receptors (TLRs) comprise a family of transmembrane proteins in plasma- and endosomal membrane of dendritic cells, phagocytes, B cells, endothelial cells, etc.: TLR-9, recognizes various microbial molecules including bacterial LPS and peptidoglycans, DNA, ssRNA, dsRNA, flagellin...

NOD-like receptor (NLRs) are found in cytoplasm of phagocytes, epithelial cells etc., NOD1/2, NALP family (inflammasomes); recognizes di-peptides and

dsDNA. RIG-I-like receptors (RLRs) – (retinoic acid-inducible gene I) in cytoplasm,

RIG-I, MDA5, LGP2; recognize viral-RNA: dsRNA and 5’-ppp-RNA and processed self-RNA.

Outside cell ->

Inside the cell ->

After recognizing PAMPs by PRR, either or both type I interferon (IFN type I) and pro-inflammatory cytokines are secreted. Upon pathogen detection, infected cells produce type I interferons. Innate immune cells, such as macrophages or dendritic cells, produce type I interferon after sensing pathogen components using various PRRs, found in cytoplasm and membranes. Plasmacytoid DCs produce large quantities of IFN-alpha. Non-immune cells, such as fibroblasts and epithelial cells, predominantly produce IFN-beta. In infected and neighboring cells type I interferons induce the expression of IFN-stimulated genes (ISGs), the products of which initiate an intracellular antimicrobial program that limit the spread of infectious agents. Innate immune cells also respond to type I interferons by enhancing antigen presentation and the production of immune response mediators, such as cytokines and chemokines. Adaptive immunity is also affected by type I interferons: e.g. type I interferons can augment antibody production by B cells and amplify the effector function of T cells. Type I interferons are protective in acute viral infections but can have either protective or deleterious roles in bacterial infections and autoimmune diseases. Innate immune response is much faster because it does not require de novo synthesis of proteins or RNA, this communication happens by cytokines or chemokines.

The functional reach of the 20-25 K human proteins is expanded by several means on three levels:

Genetic level: e.g. VDJ recombination (process by which T cells and B cells randomly assemble different gene segments – known as variable (V), diversity (D) and joining (J) genes – in order to generate unique receptors (known as antigen receptors) that can collectively recognize many different types of molecule)

RNA level: e.g. alternative splicing Protein level: e.g. protein moonlighting (moonlighting proteins comprise a

class of multifunctional proteins in which a single polypeptide chain performs multiple physiologically relevant biochemical and biophysical functions. Almost 300 proteins have been found to moonlight) & post-translational modifications

With transcriptional and post-transcriptional modifications, from 20 000 -25 000 genes we end up with more than 1 000 000 proteins. Signaling pathways are like a cascade that needs to be tightly regulated. Post-translational control relies on writer, reader, and eraser. Modified proteins can be read out only by molecules that specifically recognize the modified version of the protein. This is true for many small chemical modifiers like phosphorylation, methylations, acetylation etc. It is however true also for ubiquitin, which is actually not a small chemical modifier, but the process of ubiquitination is in essence the same as the process of phosphorylation.

UBIQUITIN

Ubiquitin is a protein, 76 amino acids long, that is activated in an ATP-dependent fashion by an E1 enzyme (ubiquitin-activating enzyme) to generate a thioester linkage between the C-terminal carboxyl group of ubiquitin and the catalytically active cysteine of the E1 enzyme, then carried by an E2 enzyme (ubiquitin-conjugation enzyme) and transferred to lysine residues on specific substrates that are recognized by specific E3 enzymes (ubiquitin-ligase enzyme). In many cases,

after the C-terminus of an ubiquitin moiety is covalently linked to a lysine residue on a target protein, the C-terminal ends (glycine residue) of subsequent ubiquitin moieties may be covalently attached to lysine residues on the preceding ubiquitin to generate a polyubiquitin chain. Shape of the polyubiquitin chains depends on what specific lysine residue on the preceding ubiquitin molecule is the site for covalent bonding (isopeptide bond) of the next ubiquitin molecule, and the shape of the chain has important functional consequences. The best understood ubiquitin polymers are Lys48-linked chains, which target proteins for proteasomal degradation, and Lys63-linked chains, which have important non-degradative functions in cell signaling, endocytosis and the DNA damage response. Lys48: when lysine of the first ubiquitin moiety is in position 48 (or 11) and forms isopeptide bonds with the C-terminus of the next ubiquitin, then the ubiquitin chain will be generated, so that is can be recognized by proteasomal cap and the protein will be targeted for degradation in the proteasome – which is a large complex of proteases that degrades ubiquitylated proteins. Once the ubiquitin is covalently linked to the lysine of the targeted protein, there are seven other exposed lysines on the ubiquitin that can covalently link another ubiquitin. Therefore there are seven different ways ubiquitin molecules can be connected and another head-to-tail option (conjugation of the ubiquitin to the amino-terminal methionine of another ubiquitin).

The writer system in ubiquitination relays on E1, E2 and E3 enzymes. In humans there are 2 E1 enzymes, 40 E2 enzymes and more than 600 E3 enzymes., meaning that E3 enzymes are the one that are responsible for the high specificity of ubiquitin. E3s are divided into three groups: homologous to E6-AP C-terminus (HECT), really interesting new gene (RING), and RING between RING (RBR). In the case of HECT E3s, ubiquitin is first transferred to the active cysteine of the E3 and subsequently the E3 adds it to the lysine of the substrate. RING and RBRs instead directly mediate transfer of ubiquitin from E2 substrates. E3 RING enzymes are not catalytically active themselves, they are more like a scaffold, but are the most abundant in humans and other mammals.

How does the ubiquitin get onto the right lysine residue of the target protein?

In lys48- or lys63-polyubiquitin chains always the same linkage type is generated. Therefore we can say that there is little or no flexibility for the ubiquitin molecules when forming chains. In

some cases, a lysine is selected through a detailed and localized biochemical mechanism (for example, in SUMOylation or ubiquitin-chain formation). In other cases, a specific lysine is selected simply through structural constraints that limit the ubiquitination machinery to acting on a given area of the target. Finally, in some cases in which the E3 ligase has a large area of action, the presence of only one or a few lysines in this target area can yield selective modification.When it comes to E3-mediated lysine specificity, there are basically three main cases: A) When E3 ligase has low lysine specificity, targeting a large area of action, also called the ubiquitination zone. B) E3 ligase can target a single lysine residue, having a limited area of action, caused by the limited structural flexibility. C) Finally, specificity can result from the availability of only one or few lysine residues on otherwise wide area of action.

Different enzymes add and remove specific ubiquitin chain types.

A particular case of lysine specificity occurs when ubiquitin itself is the target in the formation of polyubiquitin chains. A subgroup of E2s participates in specific chain formation by positioning the acceptor ubiquitin in a defined orientation to favor deposition of the donor ubiquitin on the selected lysine. The first example is the E2 E2-25K (Ube2k), which forms K48-specific chains74.

PART II

So many different receptors exist because there are many different viruses. The most abundant are RNA viruses, recognized by RIG-I.

Why does not RIG-I recognize host RNA, because we do have uncapped RNA in cytoplasm? It does not recognize host RNA, because their length, structure, 5’ end modification, and interaction with ribonucleoproteins limit the recognition. Self-RNAs therefore do not typically trigger innate immune programs of type I interferon expression.

Like RIG-I, MDA5 activates IRF3 in a cell-free system ► Both RIG-I and MDA5 CARD domains bind K63 polyubiquitin chains and activate IRF3 ► Polyubiquitin binding is required for the

activation of RIG-I and MDA5 ► Polyubiquitin binding induces the formation of a highly active RIG-I tetramer

Upon viral double-stranded RNA (dsRNA) sensing, retinoic acid-inducible gene I (RIG-I) is decorated with lysine 63 (K63)-linked ubiquitin (Ub) chains by the E3s tripartite motif containing 25 (TRIM25) and RING finger protein 135 (RNF135), and this allows its association with the mitochondria-localized adaptor protein interferon-β promoter stimulator 1 (IPS1). The recruitment of TNFR-associated factor 3 (TRAF3) to IPS1 then induces TRAF3 autoubiquitylation. The K63-

linked ubiquitin chains generated on TRAF3 facilitate activation of the IκB kinase (IKK)-related kinases TANK-binding kinase 1 (TBK1) and IKKε, leading to the subsequent phosphorylation and translocation of interferon regulatory factor 3 (IRF3) and IRF7 to the nucleus, where they drive expression of type I interferon genes. Moreover, RIG-I-dependent nuclear factor-κB (NF-κB) activation is thought to involve conjugation of K63-linked ubiquitin chains to TRAF2 and TRAF6, as this would be required in order to activate the IKK complex. In a recent study, Mao et al. suggested a model (indicated by question marks in the figure) in which cellular inhibitor of apoptosis protein 1 (cIAP1) and cIAP2 would positively regulate both the NF-κB and type I interferon responses by acting as direct E3s conjugating K63-linked ubiquitin chains to both TRAF3 and TRAF6. However, as discussed in the main text, alternative models may exist. DUBA, de-ubiquitylating enzyme A; NEMO, NF-κB essential modifier; PRR, pattern-recognition receptor.

TRIM25 induces the ubiquitination of the caspase recruitment and activation domains (CARDs). C-terminal domain (CTD) of the RIG-I is important for the initial recognition of the viral RNA.

UBIQUTIN-LIKE MOLECULES

There are many different families of ubiquitin-like molecules: molecules specific for autophagy (GABARAP, MAP1-LC3), SUMO proteins (important for translation etc.) and other. ISG15 is another type of ubiquitin-like molecules, interferon-stimulated gene 15, which is transcribed in response to interfering induction.

What does it take to be ubiquitin-like molecule?

Sequence of the ubiquitin like molecules are very different. On the other hand, ubiquitin is extremely conserved throughout species. However, all the ubiquitin-like molecules have the same secondary structure – meaning that their fold is identical to the ubiquitin molecule.

ISG15 was originally identified as an IFN-stimulated gene. This gene encodes a 15 kDa protein that has two ubiquitin-like domains with an overall sequence similarity to ubiquitin of 59.3%. and is conjugated to intracellular target proteins upon activation by interferon-alpha and interferon-beta. Several functions have been ascribed to the encoded protein, including chemotactic activity towards neutrophils, direction of ligated target proteins to intermediate filaments, cell-to-cell signaling, and antiviral activity during viral infections. The mature ISG15 polypeptide is generated from a precursor by specific cleavage of the carboxyl-terminal extension, a feature common to several ubiquitin-like proteins. Similar to conjugation of ubiquitin and other ubiquitin-like molecules, such as SUMO or NEDD8, ISG15 is ligated by an isopeptide bond to several target proteins, and when conjugated implicates several pleiotropic cellular activities. It has been reported that ISG15 is secreted by human monocytes and lymphocytes, displaying the properties of an interferon-induced cytokine. Conjugation of ISG is a reversible process and it can be reversed by de-ISGylating enzymes. (ISGylaton and de-ISGylation)

In addition to stimulation with type I IFNs, the expression of ISG15 is induced by microbial challenge, by genotoxic stress, during pregnancy and during retinoid-induced cellular differentiation. ISG15 is activated by an E1-like ubiquitin-activating enzyme (UBE1L) and is then transferred to UBCH8, which functions as an E2 for ISGylation of substrates. So far, several signaling molecules, including PLC-γ1, JAK1 and ERK, have been identified as substrates for ISG15 conjugation. Because UBCH8 is also recruited to E3 ligases, it remains unclear whether the E3 ligases that function in ubiquitin conjugation pathways and ISG15-conjugation pathways are similar or distinct.

A recent exciting development is the identification of a de-ISGylating enzyme, ubiquitin-binding protein 43 (UBP43; also known as USP18). Biochemically, UBP43 functions as a protease that specifically removes ISG15 from the proteins to which it is conjugated105. However, ablation of Ubp43 in mice leads to hypersensitivity to POLYINOSINIC:POLYCYTIDYLIC ACID (poly I:C), with reduced survival rates and decreased numbers of peripheral-blood cells and bone-marrow cells106. The JAK–STAT-signaling pathway is highly upregulated in Ubp43 –/– cells following stimulation with type I IFNs, and this is accompanied by augmented cell death. In mouse models of viral infection, it was recently found that Ubp43–/– mice are highly resistant to lethal inoculation with lymphocytic choriomeningitis virus or vesicular stomatitis virus. Viral replication in Ubp43–/– cells is abrogated, presumably as a result of the hyper-responsiveness to type I IFNs. This study shows the importance of a balance between ISGylation and de-ISGylation in the regulation of innate immune responses to viral infection. However, a more recent study showed that deficiency in ISG15 does not affect IFN-induced activation of STATs and immune responses to viral infection108. It should also be noted that UBP43 is itself regulated by ubiquitin-dependent degradation mediated by the multisubunit E3 ligase SCFSKP2, in which SKP2 is the F-box protein that recruits the substrate109, indicating that there is another layer of control, which involves ISGylation, de-ISGylation and ubiquitylation. ISG15 does not make chains.

Some ssRNA viruses edit their 5’-ppp moieties in their genomes as well as replication intermediates into 5’-monophosphate to avoid recognition by RLR. Some eukaryotes 2’-O-methylate their mRNA, allowing cellular receptors to distinguish self from unmethylated non-self mRNA through e.g. MDA5. However, flavivirus for example also started to 2’-O-methylate their RNA intermediates.

Monoubiquitination: endocytosis, membrane trafficking, histone regulation, DNA replication

Multiubiquitination: endocytosis

Polyubiquitination:

K63 polyubiquitination is induced by DNA damage and is required for DNA repair response

K48 polyubiquitination is required for proteasome-mediated degradation

RED QUEEN HYPOTHESIS postulates that parasites have to constantly evolve in order to adapt to their host species.

Type A and B influenza viruses are widespread pathogens. Type B is limited to humans and seals, whereas type A affects many species. The influenza B virus NS1 protein for instance blocks the covalent linkage of ISG15 to its target proteins by directly interacting with ISG15.