Protection by natural IgG: a sweet partnership with soluble lectins does the trick!

EMBO J 32: 2905–2919 10.1038/emboj.2013.199; published online September032013Some B cells of the adaptive immune system secrete polyreactive immunoglobulin G (IgG) in the absence of immunization or infection. Owing to its limited affinity and specificity, this natural IgG is thought to play a modest protective role. In this issue, a report reveals that natural IgG binds to microbes following their opsonization by ficolin and mannan-binding lectin (MBL), two carbohydrate receptors of the innate immune system. The interaction of natural IgG with ficolins and MBL protects against pathogenic bacteria via a complement-independent mechanism that involves IgG receptor FcγRI expressing macrophages. Thus, natural IgG enhances immunity by adopting a defensive strategy that crossovers the conventional boundaries between innate and adaptive microbial recognition systems.The adaptive immune system generates protective somatically recombined antibodies through a T cell-dependent (TD) pathway that involves follicular B cells. After recognizing antigen through the B-cell receptor (BCR), follicular B cells establish a cognate interaction with CD4⁺ T follicular helper (T_FH) cells and thereafter either rapidly differentiate into short-lived IgM-secreting plasmablasts or enter the germinal centre (GC) of lymphoid follicles to complete class switch recombination (CSR) and somatic hypermutation (SHM) (Victora and Nussenzweig, 2012). CSR from IgM to IgG, IgA and IgE generates antibodies with novel effector functions, whereas SHM provides the structural correlate for the induction of affinity maturation (Victora and Nussenzweig, 2012). Eventually, this canonical TD pathway generates long-lived bone marrow plasma cells and circulating memory B cells that produce protective class-switched antibodies capable to recognize specific antigens with high affinity (Victora and Nussenzweig, 2012).In addition to post-immune monoreactive antibodies, B cells produce pre-immune polyreactive antibodies in the absence of conventional antigenic stimulation (Ehrenstein and Notley, 2010). These natural antibodies form a vast and stable repertoire that recognizes both non-protein and protein antigens with low affinity (Ehrenstein and Notley, 2010). Natural antibodies usually emerge from a T cell-independent (TI) pathway that involves innate-like B-1 and marginal zone (MZ) B cells. These are extrafollicular B-cell subsets that rapidly differentiate into short-lived antibody-secreting plasmablasts after detecting highly conserved microbial and autologus antigens through polyreactive BCRs and nonspecific germline-encoded pattern recognition receptors (Pone et al, 2012; Cerutti et al, 2013).The most studied natural antibody is IgM, a pentameric complement-activating molecule with high avidity but low affinity for antigen (Ehrenstein and Notley, 2010). In addition to promoting the initial clearance of intruding microbes, natural IgM regulates tissue homeostasis, immunological tolerance and tumour surveillance (Ochsenbein et al, 1999; Zhou et al, 2007; Ehrenstein and Notley, 2010). Besides secreting IgM, B-1 and MZ B cells produce IgG and IgA after receiving CSR-inducing signals from dendritic cells (DCs), macrophages and neutrophils of the innate immune system (Cohen and Norins, 1966; Cerutti et al, 2013). In humans, certain natural IgG and IgA are moderately mutated and show some specificity, which may reflect the ability of human MZ B cells to undergo SHM (Cerutti et al, 2013). Yet, natural IgG and IgA are generally perceived as functionally quiescent.In this issue, Panda et al show that natural IgG bound to a broad spectrum of bacteria with high affinity by cooperating with ficolin and MBL (Panda et al, 2013), two ancestral soluble lectins of the innate immune system (Holmskov et al, 2003). This binding involved some degree of specificity, because it required the presence of ficolin or MBL on the microbial surface as well as lower pH and decreased calcium concentration in the extracellular environment as a result of infection or inflammation (see Figure 1).Open in a separate windowFigure 1Ficolins and MBL are produced by hepatocytes and various cells of the innate immune system and opsonize bacteria after recognizing conserved carbohydrates. Low pH and calcium concentrations present under infection-inflammation conditions promote the interaction of ficolin or MBL with natural IgG on the surface of bacteria. The resulting immunocomplex is efficiently phagocytosed by macrophages through FcγR1 independently of the complement protein C3, leading to the clearance of bacteria.Ficolins and MBL are soluble pattern recognition receptors that opsonize microbes after binding to glycoconjugates through distinct carbohydrate recognition domain (CRD) structures (Holmskov et al, 2003). While ficolins use a fibrinogen domain, MBL and other members of the collectin family use a C-type lectin domain attached to a collagen-like region (Holmskov et al, 2003). Similar to pentraxins, ficolins and MBL are released by innate effector cells and hepatocytes, and thus may have served as ancestral antibody-like molecules prior to the inception of the adaptive immune system (Holmskov et al, 2003; Bottazzi et al, 2010). Of note, MBL and the MBL-like complement protein C1q are recruited by natural IgM to mediate complement-dependent clearance of autologous apoptotic cells and microbes (Holmskov et al, 2003; Ehrenstein and Notley, 2010). Panda et al found that a similar lectin-dependent co-optation strategy enhances the protective properties of natural IgG (Panda et al, 2013).By using bacteria and the bacterial glycan N-acetylglicosamine, Panda et al show that natural IgG isolated from human serum or T cell-deficient mice interacted with the fibrinogen domain of microbe-associated ficolins (Panda et al, 2013). The resulting immunocomplex was phagocytosed by macrophages via the IgG receptor FcγRI in a complement-independent manner (Panda et al, 2013). The additional involvement of MBL was demonstrated by experiments showing that natural IgG retained some bacteria-binding activity in the absence of ficolins (Panda et al, 2013).Surface plasmon resonance provided some clues regarding the molecular requirements of the ficolin–IgG interaction (Panda et al, 2013), but the conformational changes required by ficolin to interact with natural IgG remain to be addressed. In particular, it is unclear what segment of the effector Fc domain of natural IgG binds to ficolins and whether Fc-associated glycans are involved in this binding. Specific glycans have been recently shown to mitigate the inflammatory properties of IgG emerging from TI responses (Hess et al, 2013) and this process could implicate ficolins and MBL. Moreover, it would be important to elucidate whether and how the antigen-binding Fab portion of natural IgG regulates its interaction with ficolins and MBL.The in vivo protective role of natural IgG was elegantly demonstrated by showing that reconstitution of IgG-deficient mice lacking the CSR-enzyme activation-induced cytidine deaminase with natural IgG from T cell-insufficient animals enhanced resistance to pathogenic Pseudomonas aeruginosa (Panda et al, 2013). This protective effect was associated with reduced production of proinflammatory cytokines, occurred independently of the complement protein C3 and was impaired by peptides capable to inhibit the binding of natural IgG to ficolin (Panda et al, 2013). Additional in vivo studies will be needed to determine whether natural IgG exerts protective activity in mice lacking ficolin, MBL or FcγRI, and to ascertain whether these molecules also enhance the protective properties of canonical or natural IgG and IgA released by bone marrow plasma cells and mucosal plasma cells, respectively.In conclusion, the findings by Panda et al show that natural IgG adopts ‘crossover'' defensive strategies that blur the conventional boundaries between the innate and adaptive immune systems. The sophisticated integration of somatically recombined and germline-encoded antigen recognition systems described in this new study shall stimulate immunologists to further explore the often underestimated protective virtues of our vast natural antibody repertoire. This effort may lead to the development of novel therapies against infections.     

RING‐between‐RINGs—keeping the safety on loaded guns

EMBO J (2012) 31 19, 3833–3844 doi:10.1038/emboj.2012.217; published online September072012EMBO Rep (2012) 13 9, 840–846 doi:10.1038/embor.2012.105; published online September072012The ‘RING-between-RING''-type E3 ubiquitin ligase HOIP acts via a novel RING/HECT-hybrid ubiquitin transfer mechanism and catalyses the formation of linear ubiquitin chains by non-covalently binding the acceptor ubiquitin. But in the absence of a binding partner, HOIP is auto-inhibited. This explains why assembly of either HOIP/HOIL-1L or HOIP/SHARPIN is required to catalyse linear chain formation.Post-translational modification of a protein with Ubiquitin (Ub) requires the activity of three enzymes: a Ub activating enzyme (E1), a Ub conjugating enzyme (E2), and a Ub ligase (E3). Final Ub transfer is performed by an E3 enzyme, which mediates the ligation of Ub from an E2∼Ub conjugate (‘∼'' denotes a thioester) onto a substrate. E3s are commonly divided into two mechanistic classes: RING/U-box E3s and HECT E3s. RING/U-box E3s facilitate the transfer of Ub from the E2∼Ub directly onto a substrate amino group. In contrast, HECTs transfer Ub from the E2∼Ub to the substrate via a HECT∼Ub intermediate. This mechanistic difference leads to an important distinction regarding what determines the type of Ub product (i.e., the specific Ub-chain linkage) formed: in ubiquitination pathways involving RING-type E3 ligases, the E2 determines the product formed, whereas for HECT-catalysed pathways, the E3 governs product formation (Christensen et al, 2007; Kim and Huibregtse, 2009).RING-between-RING (RBR) E3s comprise a class of E3s that appear to have special properties. Although RBR E3s have been considered as a subfamily of RING E3s, the RBR E3 HHARI (Human Homologue of ARIadne) was recently shown to form a HECT-like E3∼Ub intermediate (Wenzel et al, 2011). Two other members of the RBR family, HOIL-1 and HOIP, form the Linear Ub Chain Assembly Complex (LUBAC), the only E3 ligase known to catalyse the synthesis of linear Ub chains (Kirisako et al, 2006). Linear Ub chains are produced by head-to-tail conjugation of Ub molecules through their N- and C-termini and have been shown to activate the canonical NF-κB pathway (Tokunaga et al, 2009).Two studies by the Rittinger and Sixma groups now reveal important insights regarding the formation of linear Ub chains by the dimeric RBR E3 complex HOIP/HOIL-1L (Smit et al, 2012; Stieglitz et al, 2012). Results from these studies highlight three emerging themes among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions.The RBR E3 ligase domain consists of two distinct RING domains, called RING1 and RING2, connected by an IBR (In-Between-Ring) domain. Despite its name, RING2 is not a canonical RING domain as it contains an active site Cysteine (Cys), which has recently been shown to form a thioester E3∼Ub intermediate, as directly detected for the RBR E3 HHARI. Although the Ub-loaded species could not be detected for the RBR E3 parkin, mutation of the analogous cysteine residue abrogated parkin''s ligase activity implying that it works via the same mechanism. On the basis of these observations, Wenzel et al (2011) proposed that the RBR E3s are a family of RING/HECT hybrids that use RING1 to bind an E2 (RING-like) and RING2 to present the active site Cys (HECT-like) as shown schematically in Figure 1. Both Smit et al (2012) and Stieglitz et al (2012) observed a HOIP∼Ub thioester, confirming that HOIP also acts via a RING/HECT-hybrid mechanism. Furthermore, Smit et al (2012) used a clever strategy to uncouple the first transfer event (E2∼Ub to E3) from the final transfer event (E3∼Ub to substrate Ub) to verify that the E3∼Ub intermediate is a prerequisite for Ub transfer onto a substrate and not just a serendipitous side product. The results extend the number of RBR E3s for which a thioester intermediate has been observed and support the notion that RBR E3s are indeed RING/HECT hybrids.Open in a separate windowFigure 1Three common themes are emerging among RBR ligases: a RING/HECT-hybrid Ub transfer mechanism; auto-inhibition of RBR E3 activity, and a role for E3:Ub interactions. RBR E3s are characterized by their RBR domain that consists of two distinct RING domains, RING1 that binds the E2, and RING2 that harbours the active site Cys. Two new studies on the RBR E3 HOIP show that (a) domain(s) in HOIP''s N-terminal region inhibits its ligase activity and (b) a domain C-terminal to HOIP''s RBR binds and orients an acceptor Ub to direct linear Ub-chain formation (‘Linear Ub chain Determining Domain'' or LDD). (A)Three ways in which auto-inhibition might occur are illustrated: (1) inhibition of E2∼Ub binding by RING1, (2) obstruction of the active site cysteine on RING2, and/or (3) occlusion of acceptor Ub binding on the LDD. (B) A possible flow of events that occur once auto-inhibition released is shown. Details of each step and how specifically auto-inhibition is released are still unknown.Previous studies have established that HOIP Ub ligase activity and subsequent activation of NF-κB require either the RBR-containing protein, HOIL-1L, or SHARPIN, an adaptor protein associated with LUBAC (Ikeda et al, 2011; Tokunaga et al, 2011). The two current studies now show that although full-length HOIP exhibits very low activity on its own, removal of the N-terminal ∼700 residues results in robust ligase activity. Thus, HOIP appears to be auto-inhibited in the absence of a binding partner. Further analysis revealed that HOIP''s UBA (Ub-Associated) domain is partly responsible for auto-inhibition, although additional N-terminal domains appear to have auto-inhibitory effects as well. SHARPIN, which contains a UBL (Ub-Like) domain, can relieve auto-inhibition of HOIP. Similarly, the addition of the HOIL-1L UBL domain, previously shown to interact with the HOIP UBA domain (Yagi et al, 2012), relieves inhibition. Interestingly, the addition of full-length HOIL-1L results in even greater ubiquitination activity.Stieglitz et al (2012) show that the RBR E3 HOIL-1L has very low E3 activity on its own. Intriguingly, they found that mutation of the HOIL-1L RING2 active site Cys (C460A) reduced activity of the HOIP/HOIL-1L complex back to levels comparable to HOIP activity in presence of HOIL UBL alone. This suggests a more active, catalytic role for HOIL-1L in linear Ub-chain formation than previously appreciated. The details regarding this role must await further studies, but involvement of an active site Cys residue on a second RING2 domain suggests a possible reciprocal transfer mechanism. Perhaps linear chains can be pre-built via such a mechanism and passed en bloc to substrate, similarly to mechanisms used by some HECT-type bacterial E3 ligases (Levin et al, 2010).Parkin, another RBR E3, also exhibits auto-inhibition (Chaugule et al, 2011), but the auto-inhibitory mechanism and the release thereof differ from HOIP. Unlike parkin''s N-terminal UBL, which is thought to interact within the RBR domain at RING2, HOIP''s UBA does not bind detectably in trans to any region in the RBR domain (Stieglitz et al, 2012). Furthermore, addition of its UBA in trans does not inhibit the activity of HOIP RBR E3 as was seen with parkin and its UBL domain. The auto-inhibition of parkin is likely released by substrate binding, because addition of either the UIM of Eps15 or the SH3 domain of endophilin-A, both known to bind the parkin UBL, can restore the activity of parkin (Chaugule et al, 2011). In addition, phosphorylation of Ser65 within the UBL of parkin by PINK-1 activates parkin, presumably by releasing the UBL from RING2 (Kondapalli et al, 2012). In contrast, HOIP overcomes its auto-inhibition through binding either HOIL-1L or SHARPIN. There is no additive effect when both binding partners are present, consistent with the notion that both proteins act via their UBL domains, although this remains to be demonstrated for SHARPIN. The activity of either SHARPIN/HOIP or HOIL-1L/HOIP can activate NF-κB (Ikeda et al, 2011; Tokunaga et al, 2011), but how the protein complexes differ in their cellular roles remains to be further analysed.The finding that HOIP and parkin exhibit auto-inhibition raises the question whether there is something special about the RBR E3s that require auto-inhibition. In this regard, we note that RBR E3s bind the E2 UbcH7 with significantly tighter affinity than canonical RING E3s bind their E2s (Dove and Klevit, unpublished). In the absence of a substrate, RING1 loaded with UbcH7∼Ub would lead to non-productive transfer of Ub from UbcH7∼Ub to the active site of RING2. Occlusion of the active site by auto-inhibition may therefore act as a safety check until its activity is required for transfer of Ub to a substrate. As yet, there is no evidence to indicate whether substrate binding will release HOIP auto-inhibition, as it does for parkin, but this remains a possibility.The revelation that removal of all domains N-terminal to the HOIP RING1 domain yields a highly active ligase allowed both groups to explore questions pertaining to how linear chains are built. Remarkably, constructs comprised of only the RBR domain through the C-terminus of HOIP are sufficient to specify linear Ub chains. (The two groups use HOIP constructs that differ by only two N-terminal residues (697/699–1072) but Stieglitz et al call their construct RBR whereas Smit et al call it RBR-LDD.) (Smit et al, 2012; Stieglitz et al, 2012). Smit et al (2012) demonstrate that the region immediately C-terminal to RING2 is required for linear chain building activity and name the region the ‘LDD'' (Linear Ub chain Determining Domain). Their results indicate that the LDD binds and orients the acceptor Ub to promote transfer of the donor Ub from the RING2 active site to the N-terminus of the acceptor Ub (Figure 1). Parkin has also been suggested to bind free Ub. Details about whether parkin binds acceptor or donor Ub and whether Ub binding determines Ub-chain specificity are still unknown.There is precedence for acceptor Ub binding by HECT E3s and this interaction is essential for chain formation by NEDD4 and its yeast orthologue Rsp5 (Kim et al, 2011; Maspero et al, 2011). In another example, the inactive E2 variant MMS2 binds an acceptor Ub and orients the Ub-Lys63 into the active site of Ubc13 thereby guaranteeing K63-linked chain formation by the E2 (Eddins et al, 2006). Besides proper orientation of the acceptor Ub, chemical differences between α- and ɛ-amino groups likely contribute to linear Ub-chain specificity. For example, E2s known to be active with RING-type E3s can transfer Ub onto the amino acid lysine, but not the other amino acids containing α-amino groups indicating specificity towards the ɛ-amino of lysine (Wenzel et al, 2011).Catalysed by the unexpected discovery that HHARI is a HECT/RING hybrid E3, details about how the RBR class of E3s function are beginning to emerge. We now know, either directly or indirectly, that at least 4 RBR E3s of the 13 identified in humans (HHARI, HOIL, HOIP, and parkin) require a trans-thiolation event using an active site cysteine within RING2. Conservation of this cysteine among all RBR E3s strongly suggests that the RING/HECT-hybrid mechanism is conserved and therefore defines the class. The hybrid mechanism also offers an explanation for the heretofore puzzling observation that, despite being categorized as a RING E3, HOIP determines the type of Ub chain formed. The ability to bind an acceptor Ub close to the RING2 active site likely contributes to how the RBR E3s dictate the type of product they produce. Finally, both HOIP and parkin are auto-inhibited. It remains to be seen whether HOIP''s auto-inhibitory domains work via inhibition of E2∼Ub binding by RING1, obstruction of the active site cysteine on RING2, and/or occlusion of acceptor Ub binding on the LDD (Figure 1). Regardless of the mechanistic details, the ability to modulate their activity may be a common trait of the RBR E3s. Given recent rapid progress, our understanding of this special class of E3s will continue to grow apace.     

Genetic Interactions between PEROXIN12 and Other Peroxisome-Associated Ubiquitination Components

Most eukaryotic cells require peroxisomes, organelles housing fatty acid β-oxidation and other critical metabolic reactions. Peroxisomal matrix proteins carry peroxisome-targeting signals that are recognized by one of two receptors, PEX5 or PEX7, in the cytosol. After delivering the matrix proteins to the organelle, these receptors are removed from the peroxisomal membrane or matrix. Receptor retrotranslocation not only facilitates further rounds of matrix protein import but also prevents deleterious PEX5 retention in the membrane. Three peroxisome-associated ubiquitin-protein ligases in the Really Interesting New Gene (RING) family, PEX2, PEX10, and PEX12, facilitate PEX5 retrotranslocation. However, the detailed mechanism of receptor retrotranslocation remains unclear in plants. We identified an Arabidopsis (Arabidopsis thaliana) pex12 Glu-to-Lys missense allele that conferred severe peroxisomal defects, including impaired β-oxidation, inefficient matrix protein import, and decreased growth. We compared this pex12-1 mutant to other peroxisome-associated ubiquitination-related mutants and found that RING peroxin mutants displayed elevated PEX5 and PEX7 levels, supporting the involvement of RING peroxins in receptor ubiquitination in Arabidopsis. Also, we observed that disruption of any Arabidopsis RING peroxin led to decreased PEX10 levels, as seen in yeast and mammals. Peroxisomal defects were exacerbated in RING peroxin double mutants, suggesting distinct roles of individual RING peroxins. Finally, reducing function of the peroxisome-associated ubiquitin-conjugating enzyme PEX4 restored PEX10 levels and partially ameliorated the other molecular and physiological defects of the pex12-1 mutant. Future biochemical analyses will be needed to determine whether destabilization of the RING peroxin complex observed in pex12-1 stems from PEX4-dependent ubiquitination on the pex12-1 ectopic Lys residue.Oilseed plants obtain energy for germination and early development by utilizing stored fatty acids (Graham, 2008). This β-oxidation of fatty acids to acetyl-CoA occurs in peroxisomes, organelles that also house other important metabolic reactions, including the glyoxylate cycle, several steps in photorespiration, and phytohormone production (Hu et al., 2012). For example, indole-3-butyric acid (IBA) is β-oxidized into the active auxin indole-3-acetic acid (IAA) in peroxisomes (Zolman et al., 2000, 2007, 2008; Strader et al., 2010; Strader and Bartel, 2011). Many peroxisomal metabolic pathways generate reactive oxygen species (Inestrosa et al., 1979; Hu et al., 2012), and peroxisomes also house antioxidative enzymes, like catalase and ascorbate peroxidase, to detoxify hydrogen peroxide (Wang et al., 1999; Mhamdi et al., 2012).Peroxisomes can divide by fission or be synthesized de novo from the endoplasmic reticulum (ER). Preperoxisomes with peroxisomal membrane proteins bud from the ER and fuse, allowing matrix proteins to be imported to form mature peroxisomes (van der Zand et al., 2012; Mayerhofer, 2016). Peroxin (PEX) proteins facilitate peroxisome biogenesis and matrix protein import. Most peroxins are involved in importing proteins destined for the peroxisome matrix, which are imported after recognition of a type 1 or type 2 peroxisome-targeting signal (PTS). The PTS1 is a tripeptide located at the C terminus of most peroxisome-bound proteins (Gould et al., 1989; Chowdhary et al., 2012). The less common PTS2 is a nonapeptide usually located near the N terminus (Swinkels et al., 1991; Reumann, 2004). PTS1 proteins are recognized by PEX5 (van der Leij et al., 1993; Zolman et al., 2000), PTS2 proteins are recognized by PEX7 (Marzioch et al., 1994; Braverman et al., 1997; Woodward and Bartel, 2005), and PEX7 binds to PEX5 to allow matrix protein delivery in plants and mammals (Otera et al., 1998; Hayashi et al., 2005; Woodward and Bartel, 2005). The cargo-receptor complex docks with the membrane peroxins PEX13 and PEX14 (Urquhart et al., 2000; Otera et al., 2002; Woodward et al., 2014), and PEX5 assists cargo translocation into the peroxisomal matrix (Meinecke et al., 2010) before dissociating from its cargo (Freitas et al., 2011).After cargo delivery, PEX5 is recycled to enable further rounds of cargo recruitment (Thoms and Erdmann, 2006). This process requires a set of peroxins that is implicated in ubiquitinating PEX5 so that it can be retrotranslocated back to the cytosol. PEX5 ubiquitination is best understood in yeast. In Saccharomyces cerevisiae, Pex5 is monoubiquitinated through the action of the peroxisome-tethered ubiquitin-conjugating enzyme Pex4 and the peroxisomal ubiquitin-protein ligase Pex12 (Platta et al., 2009) and returned to the cytosol with the assistance of a peroxisome-tethered ATPase complex containing Pex1 and Pex6 (Grimm et al., 2012). S. cerevisiae Pex5 also can be polyubiquitinated and targeted for proteasomal degradation (Kiel et al., 2005). The cytosolic ubiquitin-conjugating enzyme Ubc4 cooperates with the peroxisomal ubiquitin-protein ligase Pex2 to polyubiquitinate Pex5 (Platta et al., 2009). Pex10 has ubiquitin-protein ligase activity (Williams et al., 2008; Platta et al., 2009; El Magraoui et al., 2012), but whether Pex10 directly ubiquitinates Pex5 is controversial. Pex10 promotes Ubc4-dependent Pex5 polyubiquitination when Pex4 is absent (Williams et al., 2008); however, Pex10 is not essential for Pex5 mono- or polyubiquitination (Platta et al., 2009), but rather enhances both Pex4/Pex12- and Ubc4/Pex2-mediated ubiquitination (El Magraoui et al., 2012). Recycling of the PTS2 receptor PEX7 is less understood, although the Pex5 recycling pathways are implicated in shuttling and degrading Pex7 in Pichia pastoris (Hagstrom et al., 2014).Although PEX5 ubiquitination has not been directly demonstrated in plants, the implicated peroxins are conserved in Arabidopsis, and several have been connected to PEX5 retrotranslocation. The PEX4 ubiquitin-conjugating enzyme binds to PEX22, which is predicted to be a peroxisomal membrane protein based on ability to restore peroxisome function to yeast mutants (Zolman et al., 2005). The pex4-1 mutant displays increased membrane-associated PEX5 (Ratzel et al., 2011; Kao and Bartel, 2015), suggesting that ubiquitin supplied by PEX4 promotes PEX5 retrotranslocation. PEX1 and PEX6 are members of the ATPases associated with diverse cellular activities (AAA) family and are tethered to peroxisomes by the peroxisomal membrane protein PEX26 (Goto et al., 2011; Li et al., 2014). The pex6-1 mutant displays PTS1 import defects and decreased PEX5 levels (Zolman and Bartel, 2004), suggesting that impaired PEX5 recycling can lead to increased PEX5 degradation. Indeed, pex4-1 restores PEX5 levels in the pex6-1 mutant (Ratzel et al., 2011), suggesting that Arabidopsis PEX4 also is involved in PEX5 ubiquitination and degradation when retrotranslocation is impeded.In addition to allowing for further rounds of PTS1 cargo import, several lines of evidence suggest that in the absence of efficient retrotranslocation, PEX5 retention in the peroxisomal membrane impairs peroxisome function. Slightly reducing levels of the PEX13 docking peroxin ameliorates the physiological defects of pex4-1 without restoring matrix protein import (Ratzel et al., 2011), presumably because decreasing PEX5 docking reduces its accumulation in the peroxisomal membrane. In addition, overexpressing PEX5 exacerbates rather than ameliorates the peroxisomal defects of pex4-1 (Kao and Bartel, 2015), suggesting that pex4-1 defects are linked to excessive PEX5 lingering in the peroxisome membrane rather than a lack of PEX5 available for import.The three Really Interesting New Gene (RING) peroxins (PEX2, PEX10, and PEX12) from Arabidopsis each possesses in vitro ubiquitin-protein ligase activity (Kaur et al., 2013). Null mutations in the RING peroxin genes confer embryo lethality in Arabidopsis (Hu et al., 2002; Schumann et al., 2003; Sparkes et al., 2003; Fan et al., 2005; Prestele et al., 2010), necessitating other approaches to study the in vivo functions of these peroxins. Expressing RING peroxins with mutations in the C-terminal zinc-binding RING domains (ΔZn) confers matrix protein import defects for PEX2-ΔZn and photorespiration defects for PEX10-ΔZn but no apparent defects for PEX12-ΔZn (Prestele et al., 2010). Targeting individual RING peroxins using RNAi confers β-oxidation deficiencies and impairs PTS1 cargo import (Fan et al., 2005; Nito et al., 2007). A screen for delayed matrix protein degradation (Burkhart et al., 2013) uncovered a missense pex2-1 mutant and a splicing pex10-2 mutant that both display PTS1 import defects (Burkhart et al., 2014), suggesting roles in regulating the PTS1 receptor, PEX5. A missense pex12 mutant (aberrant peroxisome morphology 4, apm4) has defects in β-oxidation and PTS1 import and increased membrane-associated PEX5 (Mano et al., 2006). These findings highlight the essential roles of the RING peroxins in Arabidopsis development and peroxisomal functions, but the RING peroxin interactions and the individual roles of the RING peroxins in PEX5 retrotranslocation remain incompletely understood.In this study, we describe a missense pex12-1 mutant recovered from a forward genetic screen for β-oxidation deficient mutants. The pex12-1 mutant displayed severe peroxisomal defects, including reduced growth, β-oxidation deficiencies, matrix protein import defects, and inefficient processing of PTS2 proteins. Comparing single and double mutants with impaired RING peroxins revealed that each RING peroxin contributes to complex stability and influences PEX5 accumulation. Furthermore, decreasing PEX4 function ameliorated pex12-1 defects, suggesting that the Glu-to-Lys substitution in pex12-1 lures ubiquitination, perhaps by pex12-1 itself, leading to PEX4-dependent degradation of the mutant protein.     

Stimulating cROSstalk between commensal bacteria and intestinal stem cells

EMBO J (2013) 32 23, 3017–3028 10.1038/emboj.2013.224; published online October182013Commensal gut bacteria benefit their host in many ways, for instance by aiding digestion and producing vitamins. In a new study in The EMBO Journal, Jones et al (2013) report that commensal bacteria can also promote intestinal epithelial renewal in both flies and mice. Interestingly, among commensals this effect is most specific to Lactobacilli, the friendly bacteria we use to produce cheese and yogurt. Lactobacilli stimulate NADPH oxidase (dNox/Nox1)-dependent ROS production by intestinal enterocytes and thereby activate intestinal stem cells.The human gut contains huge numbers of bacteria (∼10¹⁴/person) that play beneficial roles for our health, including digestion, building our immune system and competing with harmful microbes (Sommer and Backhed, 2013). Both commensal and pathogenic bacteria can elicit antimicrobial responses in the intestinal epithelium and also stimulate epithelial turnover (Buchon et al, 2013; Sommer and Backhed, 2013). In contrast to gut pathogens, relatively little is known about how commensal bacteria influence intestinal turnover. In a simple yet elegant study reported recently in The EMBO Journal, Jones et al (2013) show that among several different commensal bacteria tested, only Lactobacilli promoted much intestinal stem cell (ISC) proliferation, and it did so by stimulating reactive oxygen species (ROS) production. Interestingly, the specific effect of Lactobacilli was similar in both Drosophila and mice. In addition to distinguishing functional differences between species of commensals, this work suggests how the ingestion of Lactobacillus-containing probiotic supplements or food (e.g., yogurt) might support epithelial turnover and health.In both mammals and insects, ISCs give rise to intestinal enterocytes, which not only absorb nutrients from the diet but must also interact with the gut microbiota (Jiang and Edgar, 2012). The metazoan intestinal epithelium has developed conserved responses to enteric bacteria, for instance the expression of antimicrobial peptides (AMPs; Gallo and Hooper, 2012; Buchon et al, 2013), presumably to kill harmful bacteria while allowing symbiotic commensals to flourish. In addition to AMPs, intestinal epithelial cells use NADPH family oxidases to generate ROS that are used as microbicides (Lambeth and Neish, 2013). High ROS levels during enteric infections likely act non-discriminately against both commensals and pathogens, but controlled, low-level ROS can act as signalling molecules that regulate various cellular processes including proliferation (Lambeth and Neish, 2013). In flies, exposure to pathogenic Gram-negative bacteria has been reported to result in ROS (H₂O₂) production by an enzyme called dual oxidase (Duox; Ha et al, 2005). Duox activity in the fly intestine (and likely also the mammalian one) has recently been discovered to be stimulated by uracil secretion by pathogenic bacteria (Lee et al, 2013). In the mammalian intestine another enzyme, NADPH oxidase (Nox), has also been shown to produce ROS in the form of superoxide (O₂⁻), in this case in response to formylated bacterial peptides (Lambeth and Neish, 2013). A conserved role for Nox in the Drosophila intestinal epithelium had not until now been explored.Jones et al (2013) checked seven different commensal bacterial to see which would stimulate ROS production by the fly''s intestinal epithelium, and found that only one species, a Gram-positive Lactobacillus, could stimulate significant production of ROS in intestinal enterocytes. Five bacterial species were checked in mice or cultured intestinal cells, and again it was a Lactobacillus that generated the strongest ROS response. Although not all of the most prevalent enteric bacteria were assayed, those others that were—such as E. coli—induced only mild, barely detectable levels of ROS in enterocytes. Surprisingly, although bacteria pathogenic to Drosophila, like Erwinia caratovora, were expected to stimulate ROS production via Duox, Jones et al (2013) did not observe this using the ROS detecting dye hydrocyanine-Cy3, or a ROS-sensitive transgene reporter, Glutatione S-transferase-GFP, in flies. Further, Jones et al (2013) found that genetically suppressing Nox in either Drosophila or mice decreased ROS production after Lactobacillus ingestion. Consistent with the important role of Nox, Duox appeared not to be required for ROS production after Lactobacillus ingestion. In addition, Jones et al (2013) found that Lactobacilli also promoted DNA replication—a metric of cell proliferation and epithelial renewal—in the fly''s intestine, and that this was also ROS- and Nox-dependent. Again, the same relationship was found in the mouse small intestine. Together, these results suggest a conserved mechanism by which Lactobacilli can stimulate Nox-dependent ROS production in intestinal enterocytes and thereby promote ISC proliferation and enhance gut epithelial renewal.In the fly midgut, uracil produced by pathogenic bacteria can stimulate Duox-dependent ROS production, which is thought to act as a microbicide (Lee et al, 2013), and can also promote ISC proliferation (Buchon et al, 2009). However, Duox-produced ROS may also damage the intestinal epithelium itself and thereby promote epithelial regeneration indirectly through stress responses. In this disease scenario, ROS appears to be sensed by the stress-activated Jun N-terminal Kinase (JNK; Figure 1A), which can induce pro-proliferative cytokines of the Leptin/IL-6 family (Unpaireds, Upd1–3) (Buchon et al, 2009; Jiang et al, 2009). These cytokines activate JAK/STAT signalling in the ISCs, promoting their growth and proliferation, and accelerating regenerative repair of the gut epithelium (Buchon et al, 2009; Jiang et al, 2009). It is also possible, however, that low-level ROS, or specific types of ROS (e.g., H₂O₂) might induce ISC proliferation directly by acting as a signal between enterocytes and ISCs. Since commensal Lactobacillus stimulates ROS production via Nox rather than Duox, this might be a case in which a non-damaging ROS signal promotes intestinal epithelial renewal without stress signalling or a microbicidal effect (Figure 1B). However, Jones et al (2013) stopped short of ruling out a role for oxidative damage, cell death or stress signalling in the intestinal epithelium following colonization by Lactobacilli, and so these parameters must be checked in future studies. Perhaps even the friendliest symbiotes cause a bit of ‘healthy'' damage to the gut lining, stimulating it to refresh and renew. Whether damage-dependent or not, the stimulation of Drosophila ISC proliferation by commensals and pathogens alike appears to involve the same cytokine (Upd3; Buchon et al, 2009), and so some of the differences between truly pathogenic and ‘friendly'' gut microbes might be ascribed more to matters of degree than qualitative distinctions. Future studies exploring exactly how different types of ROS signals stimulate JNK activity, gut cytokine expression and epithelial renewal should be able to sort this out, and perhaps help us learn how to better manage the ecosystems in our own bellies. From the lovely examples reported by Jones et al (2013), an experimental back-and-forth between the Drosophila and mouse intestine seems an informative way to go.Open in a separate windowFigure 1Metazoan intestinal epithelial responses to commensal and pathogenic bacteria. (A) High reactive oxygen species (ROS) levels generated by dual oxidase (Duox) in response to uracil secretion by pathogenic bacteria. (B) Low ROS levels generated by NADPH oxidase (Nox) in response to commensal bacteria. In addition to acting as a microbiocide, ROS in flies may stimulate JNK signaling and cytokine (Upd 1–3) expression in enterocytes, thereby stimulating ISC proliferation and epithelial turnover or regeneration. Whether this stimulation required damage to or loss of enterocytes has yet to be explored.     

Autophagy plays a WASHing game

EMBO J (2013) 32: 2685–2696 doi:10.1038/emboj.2013.189; published online August232013Beclin 1 is a crucial regulator of autophagy. It forms a complex with ATG14L, VPS34 or the class III phosphatidylinositol 3-kinase AMBRA1 to control autophagosome formation. A study in this issue of The EMBO Journal (Xia et al, 2013) reports that WASH, a protein known to regulate endosomal sorting, hampers autophagy by inhibiting the AMBRA1-dependent polyubiquitylation of Beclin 1. This modification is required to promote VPS34 activity and to initiate autophagy during starvation.Macroautophagy (hereafter called autophagy) is a lysosomal degradation pathway for cytoplasmic components. Autophagy is initiated by the formation of a double-membrane bound vacuole, the autophagosome that sequesters the autophagic cargo and eventually fuses with the lysosomal compartment, leading to cargo degradation. Autophagy plays a major role in cytoplasm homeostasis by removing protein aggregates and controlling organelle quality, and it is stimulated to promote cell survival during stressful situations, such as nutrient starvation and microbial infection. Autophagosome formation is dependent on evolutionarily conserved Atg (Autophagy-related) proteins initially identified in yeast (Mizushima et al, 2011). They function in complexes or functional modules on a membrane known as the phagophore that matures into the autophagosome via several stages (initiation, elongation and sealing). Phosphatidylinositol 3-phosphate kinase complex I (PI3K complex I) plays a key role in the initiation step. In this complex, Beclin 1 (the mammalian homologue of yeast Atg6) interacts with ATG14L, AMBRA1, and class III PI3K or VPS34 (Cecconi and Levine, 2008; Figure 1). The activity of this complex, which produces the lipid phosphatidylinositol 3-phosphate (PI3P) to recruit Atg18 homologues (WIPIs), has to be tightly regulated to keep autophagy under control.Open in a separate windowFigure 1Role of WASH in autophagosome formation. WASH interacts with Beclin 1 via a sequence (121–221) that is not involved in endosomal sorting. In the absence of WASH, when autophagy is stimulated by nutrient deprivation, Beclin 1 interacts with VPS34 (and its adaptor VPS15), ATG14L and AMBRA1. In this complex, Beclin 1 is polyubiquitylated by AMBRA1, which acts as an E3 ligase. The activation of VPS34 produces PI3P at the phagophore to recruit WIPI proteins, and then triggers the machinery to elongate and seal the membrane, thus forming an autophagosome. The VAC domain of WASH involved in endosomal sorting is not required for autophagy regulation.In this issue of The EMBO Journal, Xia et al (2013) demonstrate that WASH (Wiskott-Aldrich syndrome protein (WASP) and SCAR homologue) regulates autophagosome formation in response to nutrient starvation by influencing the ubiquitylation of Beclin 1. WASH forms a complex with four other proteins and plays an essential role in endosomal sorting by promoting actin polymerization to facilitate segregation of endosomal proteins (Seaman et al, 2013). Using WASH−/− mice, Xia et al (2013) observe that WASH deficiency causes embryonic lethality (E7.5−E9.5) with massive apoptosis-independent cell death and the accumulation of autophagic structures. However, whether autophagy is instrumental in the cell death observed in WASH−/− embryos remains to be investigated. From a series of classical readouts, the authors conclude that WASH downregulates starvation-induced autophagy. Interestingly, WASH is detected on the autophagosomal membrane before and after closure of the autophagosome, but not on the autolysosomes (organelles formed when the autophagosome fuses with the lysosome). The authors first show that WASH has distinct roles in autophagy and in endosomal sorting, because deletion of the VCA domain of WASH that is required for its endosomal function and the knockdown of FAM21 (a protein that directs WASH to the endosomal membrane) do not influence autophagy. They then show that WASH interacts with the coil-coiled domain of Beclin 1. WASH depletion induces more VPS34 to interact with Beclin 1, leading to augmented formation of PtdIns3P and increased recruitment of WIPI-1 to the autophagosomal membrane. However, WASH does not influence the stability of Beclin 1, although it does regulate its K63-linked polyubiquitylation at position K437 during starvation (K63-linked polyubiquitylation suggests a regulatory role, whereas K48-linked polyubiquitylation is frequently associated with protein degradation). Overexpression of WASH reduces the ubiquitylation of Beclin 1, and the K437R Beclin 1 mutant that cannot be ubiquitylated has a low level of interaction with VPS34, which prevents the stimulation of autophagy by starvation (Figure 1). Recently, Beclin 1 has been shown to be ubiquitylated at position K117 during TLR4-induced autophagy (Shi and Kehrl, 2010). Interestingly, Xia et al (2013) show that the K117R Beclin 1 mutant is still ubiquitylated in response to nutrient starvation. Finally, the authors identify the substrate-specific E3 ubiquitin ligase for Beclin 1 in this context. They exclude TRAF6 and NEDD4, two E3 ligases reported to ubiquitylate Beclin 1 (Kuang et al, 2013). AMBRA1, which regulates autophagy in the PI3K complex I, is also known as DCAF3 and interacts with the Cullin 4–DDB1 E3 ligase complex (Jin et al, 2006). Following in vitro E3 ligase assays, Xia et al (2013) conclude that AMBRA1 is the substrate receptor for Beclin 1 ubiquitylation at position K437, adding Cullin4/DDB1 and AMBRA1 to the list of E3 ligases involved in autophagy (Kuang et al, 2013). The finding that WASH and AMBRA1 compete to regulate starvation-induced autophagy raises several questions: how do WASH and AMBRA1 influence one another''s binding to Beclin 1? WASH is evolutionarily conserved, although it is not present in yeast (Linardopoulou et al, 2007)—does it also reduce starvation-induced autophagy in non-mammalian cells? WASH has recently been reported to be required for the lysosomal digestion of autophagy cargo in Dictyostelium during starvation (King et al, 2013). Does this mean that the function of WASH in autophagy has evolved from controlling cargo degradation to forming autophagosomes? Xia et al (2013) do not report any association between WASH and the lysosomal compartment during autophagy; the role of WASH in autophagosome maturation calls for further investigation. Finally, a recent report demonstrates that AMBRA1 interacts with the E3 ligase TRAF6 to ubiquitylate ULK1 (the mammalian orthologue of the yeast Atg1) and to ensure its subsequent stabilization and function (Nazio et al, 2013). ULK1 is part of a complex that acts with PI3K complex 1 to regulate the initiation of autophagy (Mizushima et al, 2011). How are these two AMBRA1-dependent ubiquitylation processes coordinated to control autophagy? In conclusion, both the study reported here (Xia et al, 2013) and the recent study of Nazio et al (2013) clearly demonstrate that AMBRA1 plays a central role in regulating the initiation of autophagy.     

SUGAR-INSENSITIVE3, a RING E3 Ligase,Is a New Player in Plant Sugar Response

Sugars, such as sucrose and glucose, have been implicated in the regulation of diverse developmental events in plants and other organisms. We isolated an Arabidopsis (Arabidopsis thaliana) mutant, sugar-insensitive3 (sis3), that is resistant to the inhibitory effects of high concentrations of exogenous glucose and sucrose on early seedling development. In contrast to wild-type plants, sis3 mutants develop green, expanded cotyledons and true leaves when sown on medium containing high concentrations (e.g. 270 mm) of sucrose. Unlike some other sugar response mutants, sis3 exhibits wild-type responses to the inhibitory effects of abscisic acid and paclobutrazol, a gibberellic acid biosynthesis inhibitor, on seed germination. Map-based cloning revealed that SIS3 encodes a RING finger protein. Complementation of the sis3-2 mutant with a genomic SIS3 clone restored sugar sensitivity of sis3-2, confirming the identity of the SIS3 gene. Biochemical analyses demonstrated that SIS3 is functional in an in vitro ubiquitination assay and that the RING motif is sufficient for its activity. Our results indicate that SIS3 encodes a ubiquitin E3 ligase that is a positive regulator of sugar signaling during early seedling development.Almost all living organisms rely on the products of plant photosynthesis for sustenance, either directly or indirectly. Carbohydrates, the major photosynthates, provide both energy and carbon skeletons for fungi, plants, and animals. In addition, sugars, such as Suc and Glc, function as signaling molecules to regulate plant growth, development, gene expression, and metabolic processes. Sugar response pathways are integrated with other signaling pathways, such as those for light, phytohormones, stress, and nitrogen (Dijkwel et al., 1997; Zhou et al., 1998; Roitsch, 1999; Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Coruzzi and Zhou, 2001; Rook et al., 2001; Rolland et al., 2006).Several components of plant sugar response pathways have been identified based on the conservation of sugar-sensing mechanisms among eukaryotic cells (Rolland et al., 2001, 2006) or by mutant screens. Yeast HEXOKINASE2 functions in the Glc-mediated catabolite repression pathway (Entian, 1980). In Arabidopsis (Arabidopsis thaliana), mutations in HEXOKINASE1 (HXK1) cause a Glc-insensitive phenotype, and HXK1 demonstrates dual functions in Glc sensing and metabolism (Moore et al., 2003; Cho et al., 2006). Recent studies revealed the involvement of G-protein-coupled receptor systems in sugar response in yeast and Arabidopsis (Chen et al., 2003; Lemaire et al., 2004). Arabidopsis regulator of G-protein signaling1 (rgs1) mutant seedlings are insensitive to 6% Glc (Chen and Jones, 2004), whereas G-protein α-subunit (gpa1) null mutant seedlings are hypersensitive to Glc (Chen et al., 2003). The SNF1/AMPK/SnRK1 protein kinases are postulated to be global regulators of energy control (Polge and Thomas, 2007). Studies conducted on two members of the Arabidopsis SnRK1 (for SNF1-Related Protein Kinases1) family, AKIN10 and AKIN11, have revealed their pivotal roles in stress and sugar signaling (Baena-González et al., 2007). A genetic screen for reduced seedling growth on 175 mm Suc identified the pleiotropic regulatory locus1 (prl1) mutant, which encodes a nuclear WD protein. Further analyses revealed that PRL1 functions in Glc and phytohormone responses (Németh et al., 1998). Interestingly, PRL1 negatively regulates the Arabidopsis SnRK1s AKIN10 and AKIN11 in vitro (Bhalerao et al., 1999).Isolation of additional mutants defective in sugar response has revealed cross talk between sugar and phytohormone response pathways. For example, abscisic acid (ABA) biosynthesis and signaling mutants have been isolated by several genetic screens for seedlings with reduced responses to the inhibitory effects of high levels of Suc or Glc on seedling development. These mutants include abscisic acid-deficient1 (aba1), aba2, aba3, salt-tolerant1/nine-cis-epoxycarotenoid dioxygenase3, abscisic acid-insensitive3 (abi3), and abi4 (Arenas-Huertero et al., 2000; Huijser et al., 2000; Laby et al., 2000; Rook et al., 2001; Cheng et al., 2002; Rolland et al., 2002; Huang et al., 2008), indicating interplay between ABA- and sugar-mediated signaling. Ethylene also exhibits interactions with sugars in controlling seedling development. Both the ethylene overproduction mutant eto1 and the constitutive ethylene response mutant ctr1 exhibit Glc (Zhou et al., 1998) and Suc (Gibson et al., 2001) insensitivity, whereas the ethylene-insensitive mutants etr1, ein2, and ein4 show sugar hypersensitivity (Zhou et al., 1998; Gibson et al., 2001; Cheng et al., 2002).Further characterization of sugar response factors has suggested that ubiquitin-mediated protein degradation may play a role in sugar response. In particular, the PRL1-binding domains of SnRK1s have been shown to recruit SKP1/ASK1, a conserved SCF ubiquitin ligase subunit, as well as the α4/PAD1 proteasomal subunit, indicating a role for SnRK1s in mediating proteasomal binding of SCF ubiquitin ligases (Farrás et al., 2001). In addition, recent studies indicate that PRL1 is part of a CUL4-based E3 ligase and that AKIN10 exhibits decreased rates of degradation in prl1 than in wild-type extracts (Lee et al., 2008). The ubiquitin/26S proteasome pathway plays important roles in many cellular processes and signal transduction pathways in yeast, animals, and plants (Hochstrasser, 1996; Hershko and Ciechanover, 1998; Smalle and Vierstra, 2004). The key task of the pathway is to selectively ubiquitinate substrate proteins and target them for degradation by the 26S proteasome. In short, the multistep ubiquitination process starts with the formation of a thiol-ester linkage between ubiquitin and a ubiquitin-activating enzyme (E1). The activated ubiquitin is then transferred to a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) then mediates the covalent attachment of ubiquitin to the substrate protein. The specificity of the pathway is largely realized by the E3s, which recognize the substrates that should be ubiquitinated. In Arabidopsis, more than 1,300 genes encode putative E3 subunits and the E3 ligases can be grouped into defined families based upon the presence of HECT (for Homology to E6-AP C Terminus), RING (for Really Interesting New Gene), or U-box domains (Smalle and Vierstra, 2004). The RING-type E3s can be subdivided into single-subunit E3s, which contain the substrate recognition and RING finger domains on the same protein, and multisubunit E3s, which include the SCF (for Skp1-Cullin-F-box), CUL3-BTB (for Broad-complex, Tramtrack, Bric-a-Brac), and APC (for Anaphase-Promoting Complex) complexes (Weissman, 2001; Moon et al., 2004).The Cys-rich RING finger was first described in the early 1990s (Freemont et al., 1991). It is defined as a linear series of conserved Cys and His residues (C3HC/HC3) that bind two zinc atoms in a cross-brace arrangement. RING fingers can be divided into two types, C3HC4 (RING-HC) and C3H2C3 (RING-H2), depending on the presence of either a Cys or a His residue in the fifth position of the motif (Lovering et al., 1993; Freemont, 2000). A recent study of the RING finger ubiquitin ligase family encoded by the Arabidopsis genome resulted in the identification of 469 predicted proteins containing one or more RING domains (Stone et al., 2005). However, the in vivo biological functions of all but a few of the RING proteins remain unknown. Recent studies have implicated several Arabidopsis RING proteins in a variety biological processes, including COP1 and CIP8 (photomorphogenesis; Hardtke et al., 2002; Seo et al., 2004), SINAT5 (auxin signaling; Xie et al., 2002), ATL2 (defense signaling; Serrano and Guzman, 2004), BRH1 (brassinosteroid response; Molnár et al., 2002), RIE1 (seed development; Xu and Li, 2003), NLA (nitrogen limitation adaptation; Peng et al., 2007), HOS1 (cold response; Dong et al., 2006), AIP2 (ABA signaling; Zhang et al., 2005), KEG (ABA signaling; Stone et al., 2006), and SDIR1 (ABA signaling; Zhang et al., 2007).Here, we report the isolation, identification, and characterization of an Arabidopsis mutant, sugar-insensitive3 (sis3), which is resistant to the early seedling developmental arrest caused by high exogenous sugar levels. The responsible locus, SIS3, was identified through a map-based cloning approach and confirmed with additional T-DNA insertional mutants and complementation tests. The SIS3 gene encodes a protein with a RING-H2 domain and three putative transmembrane domains. Glutathione S-transferase (GST)-SIS3 recombinant proteins exhibit in vitro ubiquitin E3 ligase activity. Together, these results indicate that a ubiquitination pathway involving the SIS3 RING protein is required to mediate the sugar response during early seedling development.     

Cilia born out of shock and stress

EMBO J (2013) 32 23, 3029–3040 10.1038/emboj.2013.223; published online October112013Primary cilia are cell surface sensory organelles, whose dysfunction underlies various human genetic diseases collectively termed ciliopathies. A new study in The EMBO Journal by Villumsen et al now reveals how stress–response pathways converge to stimulate ciliogenesis by modulating protein composition of centriolar satellites. Better understanding of these mechanisms should bring us closer to identifying the cellular defects that underlie ciliopathies caused by mutations in centriolar satellite proteins.Centrioles are barrel-shaped structures with two distinct identities. In proliferating cells centrioles provide structural support for the centrosome, a key microtubule-organizing centre, whereas in quiescent cells centrioles are converted into basal bodies and promote the assembly of primary cilia. In centrosomes, centrioles are embedded in pericentriolar material (PCM), a dynamic structure responsible for microtubule nucleation. PCM proteins exhibit cell cycle-dependent localisation, achieved at least in part by the regulation of their transport. Centriolar satellites, dense fibrous granules frequently clustered around the interphase centrosome, have been implicated in microtubule-dependent protein transport to centrosomes (Kubo et al, 1999). In particular, PCM-1, the core constituent of centriolar satellites, is required for centrosomal accumulation of several PCM components (Dammermann and Merdes, 2002). Although the proteomic composition of satellites is still elusive, the growing list of satellite proteins includes CEP131/AZI1 (Staples et al, 2012), CEP290 (Stowe et al, 2012), Bardet-Biedl syndrome protein 4 (BBS4) and Oral facial digital syndrome protein (OFD1; Lopes et al, 2011). Mutations in OFD1, CEP290 and BBS4 cause ciliopathies (Kim et al, 2008), underscoring a functional link between satellites and ciliogenesis. So far, two roles have been proposed for satellites in cilia formation: First, in cycling cells they may serve to sequester essential ciliary proteins (Stowe et al, 2012). Second, upon initiation of the ciliogenesis programme, centriolar satellite components seem to promote the recruitment of specific ciliary proteins to basal bodies (Ferrante et al, 2006; Lopes et al, 2011; Stowe et al, 2012).In a new study in The EMBO Journal, Villumsen et al (2013) now describe how stress–response pathways conspire to control ciliogenesis. The authors observed that specific environmental stresses, such as ultraviolet light radiation (UV) or heat shock, but not ionizing radiation (IR), trigger rapid displacement of PCM-1, AZI1 and CEP290 from centriolar satellites. However, OFD1 remained associated with satellites, indicating that centriolar satellites persist despite UV-induced removal of PCM-1. This might come as some surprise, since PCM-1 depletion by RNA interference (RNAi) is thought to disrupt satellite integrity (Kim et al, 2008; Lopes et al, 2011); however, satellite loss upon PCM-1 RNAi may be a consequence of prolonged depletion of PCM-1, while acute PCM-1 displacement by stress might only ‘remodel'' centriolar satellites. It is also possible that not all satellites are created equal, and they do vary in protein composition (Kim et al, 2008; Staples et al, 2012). If so, UV-induced PCM-1 removal may disrupt some, but not all satellites.A good candidate regulator of centriolar satellite remodelling was the stress-activated MAP kinase p38, and indeed, Villumsen et al (2013) found p38 MAPK activity to be stimulated by both UV and heat shock but not IR in U2OS cells, mirroring those very stress pathways that also cause displacement of AZI1 and PCM-1 from satellites. Furthermore, p38 MAPK was essential for UV-induced dispersal of PCM-1 and AZI1. The authors then tested the hypothesis that stress-induced centriolar satellite remodelling could involve changes in the interactome of AZI1, and—consistent with an earlier proteomics study (Akimov et al, 2011)—identified PCM-1, CEP290 and the mindbomb E3 ubiquitin protein ligase 1 (MIB1) as the main AZI1 binding partners. GFP-MIB1 localized to centriolar satellites and mono-ubiquitylated AZI1, PCM-1 and CEP290 in cycling cells. In response to UV, both ubiquitylation of these proteins and MIB1 activity were reduced; notably, UV-induced MIB1 inactivation was independent of p38 MAPK activity, indicating that these two enzymes may act via distinct pathways (Figure 1A).Open in a separate windowFigure 1(A) Regulation of centriolar satellite remodelling. (B) Schematic summary of how centriolar satellite remodelling might facilitate ciliogenesis. See text for details.What could be the purpose of MIB1-dependent ubiquitylation of these satellite proteins? It certainly does not seem to regulate subcellular targeting, as in MIB1-depleted cells, AZI1 and PCM-1 both localised normally to centriolar satellites and could still be displaced by UV. Instead, ubiquitylation seems to suppress the interaction between AZI1 and PCM-1, consistent with the observation that UV, a condition that also reduces their ubiquitylation, enhances the binding of AZI1 to PCM-1.PCM-1, CEP290 and AZI1 all participate in ciliogenesis (Kim et al, 2008; Wilkinson et al, 2009; Stowe et al, 2012), raising the possibility that MIB1 might also affect this process. Indeed, serum starvation, which is known to promote cilia formation, attenuated MIB1 activity. Furthermore, MIB1 overexpression reduced the ciliogenesis observed in serum-starved cells, while MIB1 depletion in proliferating cells triggered a marked increase in the proportion of cells that formed cilia; this seems to reflect a direct effect of MIB1 on ciliogenesis, since neither MIB1 depletion nor overexpression altered cell cycle progression. Taken together, downregulation of MIB1 enzymatic activity appears to be a pre-requisite for efficient ciliogenesis, regardless of whether it is triggered by physiological ciliogenesis-promoting signals or by environmental stresses, making MIB1 a novel negative regulator of cilia formation.The recent discovery of ciliopathy-associated mutations in constituents of the DNA damage response signalling pathway pointed to a connection between DNA damage and ciliogenesis (Chaki et al, 2012). With the new link between UV and centriolar satellites, the authors next asked if UV radiation might affect ciliogenesis. Remarkably, UV and heat shock both triggered cilia assembly in RPE-1 cells in a p38 MAPK-dependent manner. MIB1 depletion further enhanced ciliogenesis after UV radiation, again implying an additive effect of p38 MAPK signalling and MIB1 suppression (Figure 1A).While finer details on the precise role of centriolar satellite components in cilia formation are still lacking, a more coherent picture is finally starting to emerge. In cycling cells, ubiquitination by MIB1 could serve to limit the interaction between AZI1 and PCM-1 on centriolar satellites (Figure 1B). Under these conditions PCM-1 may bind and sequester CEP290, an essential ciliogenic protein, thereby precluding untimely cilia formation (Stowe et al, 2012). Both during normal and stress-induced ciliogenesis programs, remodelling of centriolar satellites creates a permissive environment for cilia formation, and a key step in this process is downregulation of MIB1 activity. While it remains to be established how the latter is achieved, it is clear that MIB1 inactivation causes loss of ubiquitylation and increased binding between AZI1 and PCM-1. Preferential interaction of PCM-1 with AZI1 could in turn facilitate release of CEP290 from centriolar satellites and its subsequent accumulation at the centrosome. Once CEP290 reaches the optimum concentration at the centriole/basal body, it could serve to tether AZI1–PCM-1 complexes. PCM-1 could then concentrate Rab8 GTPase near centrosomes, allowing CEP290 to recruit Rab8 into the cilium, where it acts to extend the ciliary membrane (Kim et al, 2008).Collectively, the findings reported here provide strong experimental support to the notion that centriolar satellites are negative regulators of ciliogenesis in proliferating cells. Their role is central to limit untimely formation of cilia in cells. Environmental strains elicit stress–response pathways that converge to relieve the ciliogenesis block imposed by satellites. It is tempting to speculate that stress-induced cilia might serve as signalling platforms and contribute to checkpoint activation or perhaps initiation of repair mechanisms, but more work is needed to establish the true purpose of ciliogenesis in this context. It is of considerable interest that a recent study reports that autophagy, another stress-induced pathway, selectively removes OFD1 from satellites to promote ciliogenesis (Tang et al, 2013). Therefore stress-mediated centriolar satellite remodelling seems to be an evolving theme in the control of ciliogenesis.     

Extra-Large G Proteins Expand the Repertoire of Subunits in Arabidopsis Heterotrimeric G Protein Signaling

Heterotrimeric G proteins, consisting of Gα, Gβ, and Gγ subunits, are a conserved signal transduction mechanism in eukaryotes. However, G protein subunit numbers in diploid plant genomes are greatly reduced as compared with animals and do not correlate with the diversity of functions and phenotypes in which heterotrimeric G proteins have been implicated. In addition to GPA1, the sole canonical Arabidopsis (Arabidopsis thaliana) Gα subunit, Arabidopsis has three related proteins: the extra-large GTP-binding proteins XLG1, XLG2, and XLG3. We demonstrate that the XLGs can bind Gβγ dimers (AGB1 plus a Gγ subunit: AGG1, AGG2, or AGG3) with differing specificity in yeast (Saccharomyces cerevisiae) three-hybrid assays. Our in silico structural analysis shows that XLG3 aligns closely to the crystal structure of GPA1, and XLG3 also competes with GPA1 for Gβγ binding in yeast. We observed interaction of the XLGs with all three Gβγ dimers at the plasma membrane in planta by bimolecular fluorescence complementation. Bioinformatic and localization studies identified and confirmed nuclear localization signals in XLG2 and XLG3 and a nuclear export signal in XLG3, which may facilitate intracellular shuttling. We found that tunicamycin, salt, and glucose hypersensitivity and increased stomatal density are agb1-specific phenotypes that are not observed in gpa1 mutants but are recapitulated in xlg mutants. Thus, XLG-Gβγ heterotrimers provide additional signaling modalities for tuning plant G protein responses and increase the repertoire of G protein heterotrimer combinations from three to 12. The potential for signal partitioning and competition between the XLGs and GPA1 is a new paradigm for plant-specific cell signaling.The classical heterotrimeric G protein consists of a GDP/GTP-binding Gα subunit with GTPase activity bound to an obligate dimer formed by Gβ and Gγ subunits. In the signaling paradigm largely elucidated from mammalian systems, the plasma membrane-associated heterotrimer contains Gα in its GDP-bound form. Upon receiving a molecular signal, typically transduced by a transmembrane protein (e.g. a G protein-coupled receptor), Gα exchanges GDP for GTP and dissociates from the Gβγ dimer. Both Gα and Gβγ interact with intracellular effectors to initiate downstream signaling cascades. The intrinsic GTPase activity of Gα restores Gα to the GDP-bound form, which binds Gβγ, thereby reconstituting the heterotrimer (McCudden et al., 2005; Oldham and Hamm, 2008).Signal transduction through a heterotrimeric G protein complex is an evolutionarily conserved eukaryotic mechanism common to metazoa and plants, although there are distinct differences in the functional intricacies between the evolutionary branches (Jones et al., 2011a, 2011b; Bradford et al., 2013). The numbers of each subunit encoded within genomes, and therefore the potential for combinatorial complexity within the heterotrimer, is one of the most striking differences between plants and animals. For example, the human genome encodes 23 Gα (encoded by 16 genes), five Gβ, and 12 Gγ subunits (Hurowitz et al., 2000; McCudden et al., 2005; Birnbaumer, 2007). The Arabidopsis (Arabidopsis thaliana) genome, however, only encodes one canonical Gα (GPA1; Ma et al., 1990), one Gβ (AGB1; Weiss et al., 1994), and three Gγ (AGG1, AGG2, and AGG3) subunits (Mason and Botella, 2000, 2001; Chakravorty et al., 2011), while the rice (Oryza sativa) genome encodes one Gα (Ishikawa et al., 1995), one Gβ (Ishikawa et al., 1996), and either four or five Gγ subunits (Kato et al., 2004; Chakravorty et al., 2011; Botella, 2012). As expected, genomes of polyploid plants have more copies due to genome duplication, with the soybean (Glycine max) genome encoding four Gα, four Gβ (Bisht et al., 2011), and 10 Gγ subunits (Choudhury et al., 2011). However, Arabidopsis heterotrimeric G proteins have been implicated in a surprisingly large number of phenotypes, which is seemingly contradictory given the relative scarcity of subunits. Arabidopsis G proteins have been implicated in cell division (Ullah et al., 2001; Chen et al., 2006) and morphological development in various tissues, including hypocotyls (Ullah et al., 2001, 2003), roots (Ullah et al., 2003; Chen et al., 2006; Li et al., 2012), leaves (Lease et al., 2001; Ullah et al., 2001), inflorescences (Ullah et al., 2003), and flowers and siliques (Lease et al., 2001), as well as in pathogen responses (Llorente et al., 2005; Trusov et al., 2006; Cheng et al., 2015), regulation of stomatal movement (Wang et al., 2001; Coursol et al., 2003; Fan et al., 2008) and development (Zhang et al., 2008; Nilson and Assmann, 2010), cell wall composition (Delgado-Cerezo et al., 2012), responses to various light stimuli (Warpeha et al., 2007; Botto et al., 2009), responses to multiple abiotic stimuli (Huang et al., 2006; Pandey et al., 2006; Trusov et al., 2007; Zhang et al., 2008; Colaneri et al., 2014), responses to various hormones during germination (Ullah et al., 2002), and postgermination development (Ullah et al., 2002; Pandey et al., 2006; Trusov et al., 2007). Since the Gγ subunit appeared to be the only subunit that provides diversity in heterotrimer composition in Arabidopsis, it was proposed that all functional specificity in heterotrimeric G protein signaling was provided by the Gγ subunit (Trusov et al., 2007; Chakravorty et al., 2011; Thung et al., 2012, 2013). This allowed for only three heterotrimer combinations to account for the wide range of G protein-associated phenotypes.In addition to the above typical G protein subunits, the plant kingdom contains a conserved protein family of extra-large GTP-binding proteins (XLGs). XLGs differ from typical Gα subunits in that they possess a long N-terminal extension of unknown function, but they are similar in that they all have a typical C-terminal Gα-like region, with five semiconserved G-box (G1–G5) motifs. The XLGs also possess the two sequence features that differentiate heterotrimeric G protein Gα subunits from monomeric G proteins: a helical region between the G1 and G2 motifs and an Asp/Glu-rich loop between the G3 and G4 motifs (Lee and Assmann, 1999; Ding et al., 2008; Heo et al., 2012). The Arabidopsis XLG family comprises XLG1, XLG2, and XLG3, and all three have demonstrated GTP-binding and GTPase activities, although they differ from GPA1 in exhibiting a much slower rate of GTP hydrolysis, with a Ca²⁺ cofactor requirement instead of an Mg²⁺ requirement, as for canonical Gα proteins (Heo et al., 2012). All three Arabidopsis XLGs were observed to be nuclear localized (Ding et al., 2008). Although much less is known about XLGs than canonical Gα subunits, XLG2 positively regulates resistance to the bacterial pathogen Pseudomonas syringae and was immunoprecipitated with AGB1 from tissue infected with P. syringae (Zhu et al., 2009). xlg3 mutants, like agb1 mutants, are impaired in root-waving and root-skewing responses (Pandey et al., 2008). During the preparation of this report, Maruta et al. (2015) further investigated XLG2, particularly focusing on the link between XLG2 and Gβγ in pathogen responses. Based on symptom progression in xlg mutants, they found that XLG2 is a positive regulator of resistance to both bacterial and fungal pathogens, with a minor contribution from XLG3 in resistance to Fusarium oxysporum. XLG2 and XLG3 are also positive regulators of reactive oxygen species (ROS) production in response to pathogen-associated molecular pattern elicitors. The resistance and pathogen-associated molecular pattern-induced ROS phenotypes of the agg1 agg2 and xlg2 xlg3 double mutants were not additive in an agg1 agg2 xlg2 xlg3 quadruple mutant, indicating that these two XLGs and the two Gγ subunits function in the same, rather than parallel, pathways. Unfortunately, the close proximity of XLG2 and AGB1 on chromosome 4 precluded the generation of an agb1 xlg2 double mutant; therefore, direct genetic evidence of XLG2 and AGB1 interaction is still lacking, but physical interactions between XLG2 and the Gβγ dimers were shown by yeast (Saccharomyces cerevisiae) three-hybrid and bimolecular fluorescence complementation (BiFC) assays (Maruta et al., 2015). Localization of all three XLGs was also reexamined, indicating that XLGs are capable of localizing to the plasma membrane in addition to the nucleus (Maruta et al., 2015).Interestingly, several other plant G protein-related phenotypes, in addition to pathogen resistance, have been observed only in Gβ and Gγ mutants, with opposite phenotypes observed in Gα (gpa1) mutants. Traditionally, the observation of opposite phenotypes in Gα versus Gβγ mutants in plants and other organisms has mechanistically been attributed to signaling mediated by free Gβγ, which increases in abundance in the absence of Gα. However, an intriguing alternative is that XLG proteins fulfill a Gα-like role in forming heterotrimeric complexes with Gβγ and function in non-GPA1-based G protein signaling processes. If XLGs function like Gα subunits, the corresponding increase in subunit diversity could potentially account for the diversity of G protein phenotypes. In light of this possibility, we assessed the heterotrimerization potential of all possible XLG and Gβγ dimer combinations, XLG localization and its regulation by Gβγ, and the effect of xlg mutation on selected known phenotypes associated with heterotrimeric G proteins. Our results provide compelling evidence for the formation of XLG-Gβγ heterotrimers and reveal that plant G protein signaling is substantially more complex than previously thought.