Remodeling Without Destruction: Non-proteolytic Ubiquitin Chains In .
Molecular Psychiatry (2021) 9-7EXPERT REVIEWRemodeling without destruction: non-proteolytic ubiquitin chainsin neural function and brain disordersAlexis Zajicek1 Wei-Dong Yao 11234567890();,:1234567890();,:Received: 17 January 2020 / Revised: 8 July 2020 / Accepted: 14 July 2020 / Published online: 24 July 2020 Springer Nature Limited 2020AbstractUbiquitination is a fundamental posttranslational protein modification that regulates diverse biological processes, includingthose in the CNS. Several topologically and functionally distinct polyubiquitin chains can be assembled on proteinsubstrates, modifying their fates. The classical and most prevalent polyubiquitin chains are those that tag a substrate to theproteasome for degradation, which has been established as a major mechanism driving neural circuit deconstruction andremodeling. In contrast, proteasome-independent non-proteolytic polyubiquitin chains regulate protein scaffolding, signalingcomplex formation, and kinase activation, and play essential roles in an array of signal transduction processes. Despite beinga cornerstone in immune signaling and abundant in the mammalian brain, these non-proteolytic chains are underappreciatedin neurons and synapses in the brain. Emerging studies have begun to generate exciting insights about some fundamentalroles played by these non-degradative chains in neuronal function and plasticity. In addition, their roles in a number of braindiseases are being recognized. In this article, we discuss recent advances on these nonconventional ubiquitin chains in neuraldevelopment, function, plasticity, and related pathologies.IntroductionUbiquitination is an essential posttranslational proteinmodification in eukaryotes through which the 76 amino acidprotein, ubiquitin is conjugated to a protein substrate [1].Ubiquitination is completed in sequential enzymatic eventsthrough E1 ubiquitin-activating, E2 ubiquitin-conjugating,E3 ubiquitin-ligating enzymes, and sometimes an E4 ligase,and is reversed by a family of deubiquitinases (DUBs)(Fig. 1a). A major form of ubiquitination is polyubiquitination where a chain of ubiquitin molecules linkedthrough the C-terminal glycine of one ubiquitin moleculeand an internal lysine of another ubiquitin molecule isconjugated on a lysine residue of a substrate. Given thatseven internal lysine residues (K6, K11, K27, K29, K33,K48, and K63) exist in the ubiquitin molecule, seven distinct polyubiquitin (polyUb) chains can form [2] (Fig. 1b).* Wei-Dong Yaoyaow@upstate.edu1Departments of Psychiatry & Behavioral Sciences, and ofNeuroscience & Physiology, State University of New YorkUpstate Medical University, Syracuse, NY 13210, USAAnother polyUb, the head-to-tail linear chain (M1) linkedthrough the N-terminal methionine (M) of one ubiquitin andthe C terminus of a preceding ubiquitin also exists [3].Various polyUb chains assume distinct topologies andconfer differential fates to a substrate. K48-linked chains arethe classical signal that targets substrates to the proteasomesfor degradation, whereas K63 and M1 chains are nonproteolytic and are involved in signaling activation andtransduction [4]. The remaining chains, less prevalent andatypical, have both proteasome-dependent and nonproteolytic functions.The roles of the ubiquitin–proteasome system (UPS) inneural development, function, plasticity, and behavior havebeen extensively investigated [5–8]. Notably, UPSdependent protein degradation and turnover represent amajor mechanism driving synapse remodeling and plasticity, and are involved in a number of brain diseases [7–10].Non-proteolytic ubiquitination, however, has not been wellstudied in neurons. Recent studies begin to reveal involvements of non-proteolytic polyUb chains in neural development, receptor trafficking, pre- and postsynaptic functionand remodeling, and synaptic plasticity, representing a newparadigm in synapse biology. The potential roles of nonproteolytic chains in several common brain diseases,including autism spectrum disorders (ASDs), schizophrenia,
248A. Zajicek, W.-D. YaoFig. 1 Different faces ofpolyubiquitin linkages.a Schematic of enzymatic stepsinvolved in substrateubiquitination anddeubiquitination. Ubiquitinationby E1, E2, and E3 enzymes, aswell as ubiquitin chain extensionby U-Box/E4 enzymes anddeubiquitination bydeubiquitinases (DUB) areshown. Mechanisms for RING,HECT, and RBR E3 ligases aredepicted. IBR in between ring,SBR substrate binding region.b Eight different chaintopologies and their degradativeand non-degradative roles inprotein fates are shown. TNFtumor necrosis factor, ERADendoplasmic reticulumassociated degradation, Mmethionine, K lysine, G glycine.Parkinson’s disease (PD), Alzheimer’s Disease (AD), andrelated dementia, have also been steadily emerging.In this review, we discuss recent advances on the roles ofnon-proteolytic polyubiquitination in neural development,functions, and plasticity, as well as several common braindisorders. Although not involved in proteasomal degradation,K63-polyUb chains can target ubiquitinated cargos, includingmisfolded protein inclusions (aggrephagy) and damagedmitochondria (mitophagy) to the autophagy–lysosome pathway for clearance. This proteasome-independent proteolysisfunction is outside the scope of our discussion, and interestedreaders may refer to some excellent recent reviews on thistopic [11–14].Different faces of ubiquitin linkagesAmong the eight polyUb linkages (Fig. 1b), K48 chains arethe first identified and best characterized. These chainsadopt a compact conformation and are recognized by the26S proteasome, where substrates are degraded and theubiquitin tag is cleaved and recycled, and act as a generaldegradation signal maintaining cellular protein homeostasis[2, 15]. K63 chains represent the second most abundantpolyUb species in the mammalian brain [16]. They adopt amore open and flexible conformation and are not recognizedby the 26S proteasome, thus do not signal proteasomaldegradation. Instead, they mediate signaling pathwaysinvolved in NF-κB activation, receptor endocytosis, andDNA repair, by stabilizing/activating substrates or acting asa scaffold to assemble signaling complexes [2, 4, 17, 18].Linear chains are also non-degradable by the proteasomeand play important roles in NF-κB signaling [3, 19]. Theremaining linkages are less prevalent and play degradativeand/or non-degradative roles in more specialized cellularprocesses [4, 20]. K6 chains are implicated in DNA damageresponse (non degradative) and mitophagy (degradative).K11 chains regulate the proteasomal degradation of proteinsinvolved in cell cycle regulation [21, 22] and endoplasmicreticulum-associated degradation [23], and have a nonproteolytic signaling role in TNFα activation [24]. K27chains are more enigmatic, but are implicated in DNAdamage, mitophagy, and autophagy as scaffolds to recruitsignaling proteins [20]. Both K29 and K33 chains can serveas proteasome degradation signals and are implicated inepigenetic regulation and post-Golgi membrane proteintrafficking as non-proteolytic signals [20, 25, 26]. Finally,mixed/branched and unanchored chains also exist and playdegradative and non-degradative functions [20, 27].The various polyUb linkages are assembled by diverse( 600) E3s in complex with 40 E2s [2, 28, 29]. E3s containfour families: really interesting new gene (RING), homologousto E6-associated protein C-terminus (HECT), UFD2 homology (U-box), and RING-in-between-RING (RBR) [30, 31](Fig. 1a). For HECT E3s, ubiquitin is relayed from a bound E2to a catalytic cysteine residue in the HECT domain of an E3,which then attaches the ubiquitin to a substrate. The chain
Remodeling without destruction: non-proteolytic ubiquitin chains in neural function and brain disordersspecificity for most HECT E3s remains to be determined, butone exception is Nedd4 (neural precursor cell expresseddevelopmentally downregulated gene 4) that contains a Ubbinding site in the C lobe which may orient the Ub chain andselect for K63 chains [32]. In contrast, RING E3s, the mostprevalent, serve as adaptors, bringing a ubiquitin-bound E2and a substrate into close proximity and activating the E2 totransfer the ubiquitin to the substrate. Thus, E2s bound to theseRING E3s often determine the polyUb linkage specificity. TheRING E3 ligase, TNF receptor-associated factor 6 (TRAF6),for example, catalyzes the formation of K63 chains on substrates together with the K63-specific ubiquitin-conjugatingenzyme Ubc13 and the Ubc-like protein Uev1A E2 dimers[33]. Some RING E3s function as single subunits (e.g.,TRAFs), whereas others function in multisubunit complexesthat contain several adaptors for substrate recognition andubiquitin E3 activity (e.g., Skp1/Cullin/F-Box (SCF)) [34]. Ubox E3s, also dubbed E4s, mainly elongate existing polyUbchains [35]. Finally, RBR E3s employ a hybrid mechanism bywhich the first RING domain acts as a canonical RING ligasewhile the second RING domain acts like a HECT ligase[36, 37]. Two important RBR E3s are Parkin, involved in PDand mitophagy [13], and LUBAC (linear ubiquitin chainassembly complex), an E3 ligase complex composed ofSHARPIN, HOIL-IL, and HOIP that specifically generateslinear chains [3]. All ubiquitin enzymes discussed in thisreview are summarized in Table 1.More than 100 DUBs, cysteine proteases and metalloproteases that remove or trim PolyUb chains, exist in thehuman genome [38–40]. Based on their catalytic domainsand mechanisms of action, these DUBs are categorized intosix superfamilies: Ubiquitin C-terminal hydrolases (UCHs),ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), the Josephin family, the Motif interactingwith ubiquitin-containing novel DUB family (MINDYs),and the JAB1/MPN/MOV34 (JAMMs) metalloproteasefamily [38, 40, 41]. DUB superfamilies exhibit variablelevels of linkage specificity. For example, USPs are typically linkage nonspecific [38], but an exception is cylindromatosis (CYLD), which is specific to K63 (and M1chains which have virtually equivalent structure) due to itsunique UBD that contains an extended loop near the catalytic domain selective for K63 chains [42, 43]. In contrast,OTUs are mostly linkage-specific due to their diverseubiquitin-binding domains (UBDs) [44].Diverse polyUb chains are “decoded” by a large numberof UBD-containing proteins, known as ubiquitin receptors,which translate the ubiquitin code to specific biochemicaland cellular outputs [2, 45]. There are more than 20 structurally different UBDs in five subfamilies, αHelix, ZincFinger (ZnF), Ubc-like, pleckstrin homology (PH) fold, andother structures [2]. Individual UBDs typically bind ubiquitin with low affinities, and additional interactions249between ubiquitinated targets and ubiquitin receptors in amultiprotein complex cooperate to provide sufficient specificity, avidity, and dynamic regulation. The formation ofthese “signalosomes” through recruitment of binding partners that harbor specific UBDs is a hallmark role of nondegradative polyUb chains.Major non-proteolytic paradigms in non neuronsSignalosome assembly and kinase activationRoles of non-proteolytic polyUb in scaffolding signalingcomplexes and activating kinases are best illuminated in theclassical NF-κB signaling pathways in innate and adaptiveimmunity (Fig. 2a) [18, 46–48]. Activation of Toll-like andcytokine receptors recruits, in a ligand- and receptorspecific manner, adaptor proteins, protein kinases, and apanel of K63- or M1-specific E3 ligases including TRAF6and LUBAC, promoting their activation. Activated E3scoordinate with specific E2s to assemble K63 and M1chains on specific substrates or free, unanchored chains.These polyUb chains then serve as scaffolds to simultaneously load the TAK1 (TGF-β activated kinase 1) kinasecomplex and IKK (Inhibitor-of-κB kinase) complex throughtheir UBDs, leading to activation of the IKK complex.Activated IKK complex then recruits a signaling complexconsisting of IkBα (inhibitor-of NF-κB) and NF-κB subunits p65/p50, leading to phosphorylation and proteasomaldegradation of IkBα by the E3 complex SCFβTrCP [49].Liberated NF-κB then enters the nucleus to regulateexpression of diverse target genes important for inflammation and cell survival. The ubiquitin-editing enzyme A20,an OTU DUB, and CYLD are potent inhibitors of NF-κBsignaling by cleaving K63 and M1 chains [19, 50].EndocytosisUbiquitination is a classical signal for surface receptorendocytosis, endosomal trafficking, and sorting [17, 51, 52].Upon ligand binding, activated receptors are ubiquitinated atthe plasma membrane by several K63 E3 ligases recruited tothe activated receptors, such as CBL (Cas-Br-M ecotropicretroviral transforming sequence; a RING E3), TRAF6, andthe Nedd4 family. AMSH (associated molecule with the SH3domain of STAM; a JAMM DUB) and USP8/UBPY (ubiquitin-specific protease 8/ubiquitin-specific protease Y)negatively regulate endocytosis by cleaving K63 chains frominternalized receptors [53, 54]. Ubiquitinated receptors areinternalized through either clathrin-dependent or -independentendocytosis, driven by a set of endocytic adaptors at theplasma membrane, such as EPS15 (epidermal growth factorreceptor substrate 15) and epsins, which bind ubiquitinthrough their UBDs and couple receptors to endocytic
250A. Zajicek, W.-D. YaoTable 1 Ubiquitin enzymes and their characteristics discussed in this article.Ubiquitin enzyme Enzyme type ChainKnown substratesspecificityNeuronal functionRefs.UBA6Ubc13E1E2N/AK63NDTRAF6, H2A/H2AX[158][33, 56–58, 93, NG E3RING E3K27, K63K63K48K63K63K6, othersK63HERC2LUBACHECT E3RBR E3complexHECT E3K63M1Synapse formationUnknown[89][3, 19]HECT E3K63AMPAR trafficking; homeostatic plasticity; αsynuclein endosomal trafficking and clearanceAMPAR trafficking; homeostatic plasticity[91, 115, 126, 127, 192, or tyrosine kinases,e.g., EGFRNDNEMO, MyD88,IRAKs, TNFR1GluA1, mGluR7, αsynucleinGluA1UnknownSynapse formation; PSD-95 ubiquitination andscaffolding; locomotor and plasticity behaviorUnknownNeurotrophin signalingUnknownUnknownPSD-95 ubiquitination and scaffoldingUnknownUnknownRBR INGNedd4CYLDK63H2A/H2AXK63WASHNDGluA2K63, K27 H2A/H2AXK63GlisK11, K48 GlisK48IκBαK63NDK63PSD-95, TrkA, TrkB,TrkC, p75, NRIF, Ulk1/2,presenilinsOTUK48, K63 RIP1, TRAF6,DUB, E3NEMO, Ubc13JAMM DUB K63Receptor tyrosine kinases,e.g., EGFRUSP DUBK63, M1 PSD-95USP7USP8/UBPYUSP DUBUSP DUBUSP46USP DUBA20AMSHE3E3E3E3E3E3E3E3E3K63TDP-43 cytoplasmic translocation and accumulation inFTD/ALSSynapse formationEndosomal actin levels and protein recyclingAMPAR trafficking, homeostatic plasticityNDNeural developmentNeural developmentNF-κB signalingPresent in the PSD with unknown functionNeurotrophin signaling; synapse remodeling; axonaloutgrowth[58, 59][63][89][158][33, 93][56–60][51, 52][129][183][56, 57, 89][152, 153][117][56–58][78][74–77][49][94][33, 63–71, 73, 81, 93, 172]Synapse remodeling[50, 95]Unknown[53]Disassembles K63 chains in PSD; regulates PSDscaffolding; cLTD; striatal GABAergic transmission;axonal outgrowthK48, K63 WASHEndosomal actin levels and protein recyclingK63GluA1, α-synuclein, EGFR AMPAR trafficking; homeostatic plasticity; αsynuclein clearance and toxicityK63GLR-1, GluA1, GluA2AMPAR trafficking[42–44, 93, 100–102, 104–106][150, 152][54, 127, 191][118, 130]ALS amyotrophic lateral sclerosis, AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor, cLTD chemical long-termdepression, FTD frontotemporal dementia, GABA gamma-aminobutyric acid, N/A not applicable, ND not discussed, PSD postsynaptic density.vesicles. Once at early endosomes, ubiquitinated cargo issorted through endosome-associated complexes ESCRT(endosomal sorting complex required for transport) 0, -I, -II,and -III into multivesicular bodies (MVBs) withinlate endosomes for degradation (Fig. 2b). Importantly,each ESCRT complex contains UBDs in their subunits thatfacilitate formation of multiprotein complexes necessary forefficient endosomal sorting. Ubiquitin is removed by ESCRTIII-associated DUBs prior to receptor entry into MVBs.Monoubiquitin and K63-polyUb chains often dominate inendocytosis, however, the latter is believed to be moreeffective [55].Maintenance of genome integrityNon-proteolytic polyUb plays essential roles in DNAdamage response (Fig. 2c). Following DNA doublestranded breaks (DSBs), the E2 Ubc13 and UbcH5c, andRING E3 ligases RNF (RING finger protein) 8 (RNF8) andRNF168 are recruited to sites of damage foci, where theysynthesize K27 and K63 chains on Histone H2A/H2AX orother substrates [56–59]. These non-proteolytic chains serveas scaffolds for the receptor-associated protein 80 (RAP80),a ubiquitin receptor, which recruits the BRCA1 (breastcancer type 1 susceptibility protein) E3 ligase repair
Remodeling without destruction: non-proteolytic ubiquitin chains in neural function and brain disorders251Fig. 2 Roles of non-proteolytic polyUb in signalosome formation,kinase activation, and genome integrity. a NF-κB signaling. Activation of TNF receptor (TNFR), interleukin 1 receptor (IL-1R), orToll-like receptor (TLR) leads to, in a receptor-dependent manner,recruitment of adaptor proteins (TRADD, MyD88), protein kinases(RIP1, IRAKs), and E3 ubiquitin ligases (TRAF2/5/6, cIAP1/2, andLUBAC) which, together with specific E2s (not shown), catalyze thesynthesis of anchored or unanchored polyUb chains of different linkages (K63, M1, and K11). These non-proteolytic chains bindTAB2 subunit of the TAK1 kinase complex, resulting in TAK1 activation, which then phosphorylates IKKβ of the IKK complex, leadingto IKK activation. Activated IKK phosphorylates IκB proteins,resulting in their K48-linked ubiquitination and subsequent degradation by the proteasome, freeing the NF-κB complex to enter thenucleus to regulate gene transcription. CYLD and A20 inhibit NF-κBsignaling by cleaving K63 and M1 chains. b Endocytosis. Followingligand binding of the receptor tyrosine kinases EGFR on the plasmamembrane, the E3 CBL conjugates K63 chains onto the activatedreceptors. DUBs, such as AMSH and USP8/UBPY, cleave K63 chainsfrom internalized receptors. Endocytic adaptors EPS15 and Epsinrecognize ubiquitin-bound receptors, targeting them for sorting by theendosome. The ESCRT-0, -I, and -II complexes mediate the sortingprocess and target the receptors for ESCRT-III dependent lysosomaldegradation, or deubiquitination by DUBs (asterisk (*) indicates largely unknown) and recycling back to the plasma membrane.c Maintenance of genome integrity. Following DNA double-strandedbreaks (DSBs), the E3/E2 complexes RNF8/Ubc13 and RNF168/UbcH5c are recruited to histone H2A/H2AX at sites of DNA damage,where they synthesize K63 and K27 chains on histones or othersubstrates (S). RAP80 then binds to the polyUb chains, recruiting theBRCA1 E3 ligase complex to sites of the DSB, where it synthesizesK6 chains on substrates to initiate DNA repair process.complex and other crucial mediators to DSB-associatedchromatin to promote K6 ubiquitination and initiate DNArepair [56–60].Neurotrophin signalingNeurotrophins (NGF, BDNF, NT4/5, and NT-3) regulateneuronal differentiation, survival, and plasticity bysignaling through several tyrosine kinase receptors (TrkA,TrkB, and TrkC) and the p75 receptor, a member of theTNF receptor superfamily [61, 62]. The NGF receptor TrkAundergoes K63 polyubiquitination following NGF stimulation in PC12 cells [63], which is mediated by TRAF6 andthe E2 UbcH7, and requires the K63-polyUb-bindingscaffolding protein p62/sequestosome-1 (SQSTM1) tofacilitate the assembly of NGF-induced p75-TrkA-TRAF6UbcH7 multiprotein complex. This K63-polyUb signal isrequired for TrkA internalization, downstream signaling,and NGF-induced neurite outgrowth [63]. TrkB and TrkCalso contain a consensus site for TRAF6/p62 polyubiquitination, and TrkB is ubiquitinated by TRAF6 following its activation [64].TRAF6 becomes associated with p75 following stimulation by neurotrophins in transfected HEK293T cells [65].TRAF6 may act as the E3 ligase for p75, which is polyubiquitinated following NGF stimulation in a mouse hippocampal cell line [66]. Additional signaling intermediatesrecruited to activated p75 or TRAF6 include p62 [67],IRAK (interleukin 1 receptor-associated kinase) [68], NRIF(neurotrophin receptor-interacting factor) [69, 70], andRIP2 (receptor-interacting protein 2) [71]. Among these,NRIF is K63 ubiquitinated by TRAF6 [70]. These cytoplasmic adaptors form complexes following p75 activation
252A. Zajicek, W.-D. YaoFig. 3 Non-proteolytic ubiquitination in synapse development andremodeling. Schematic of K63 ubiquitination-related molecularpathways involved in pre- and postsynaptic development and/orremodeling. Presynaptically, the ubiquitin adaptor protein PLAA isrequired for sorting of ubiquitin-modified membrane proteins into thelumen of MVB/late endosomes. This process plays an important rolein synaptic vesicle (SV) recycling, reserve pool size, synaptic transmission, and terminal differentiation. K63 ubiquitination also regulatesfilopodia extension and branching of sensory axons via an endocyticprocess. NGF induces K63-polyubiquitination of Ulk1/2 likely byTRAF6, allowing the binding of Ulk1/2 to p62 and the recruitment ofUlk1/2 to active TrkA complex via p62, leading to TrkA internalization, attenuation of NGF signaling, and filopodia withdrawal. RNF8/HERC2 may also regulate presynaptic differentiation in a K63dependent manner through ubiquitination of unidentified substrates(S). A lysosome, although primarily localized in the soma, is shownhere to facilitate illustration and discussion. Postsynaptically, A20inhibits dendritic arborization and spine remodeling through suppression of NF-κB activity, likely via regulation of gene expression in thenucleus, causing postsynaptic remodeling. TRAF6 and CYLD controllocal postsynaptic remodeling through K63-polyUb chain conjugationand disassembly on PSD-95. PSD-95 is constitutively K63ubiquitinated by TRAF6 in conjunction with Ubc13/Uev1A, anddeubiquitinated by CYLD. The K63-polyUb conjugation sites arelocalized primarily in the GK domain, where the assembled K63chains are essential for PSD-95 interactions with its GK bindingpartners including SPAR, GKAP/SAPAP, and AKAP79/150. K63ubiquitinated PSD-95, but not its un-ubiquitinated form, is targeted tothe PSD to promote synapse efficacy (AMPAR numbers) andmaturation. CYLD removes K63-polyUb from PSD-95, leading totranslocation of deubiquitinated PSD-95 away from the PSD, destabilizing the PSD, weakening the synapse, and inhibiting synapseformation and maturation. PDZ, SH3, and GK domains of PSD-95 areindicated. PSD-95 binds NMDARs directly and AMPARs throughStargazin and localizes these receptors at the synapse.and transduce multiple downstream p75-dependentsignaling pathways to regulate cell survival orapoptosis in various neuron(-like) cell types [65, 66, 68, 70–72]. In TRAF6 knockout mice, p75-mediated NF-κBand JNK (c-Jun N-terminal kinase) signaling followingNGF or BDNF stimulation are blunted and the p75-inducedapoptosis is lost in Schwann cells and sympathetic neurons[73].K63 chains on all Glis, either in their activator or repressorforms, and the K63 chains promote Gli nuclear export andrefine their gradients by limiting their nuclear levels. Lossof RNF220 leads to expansion of the GliA and GliR gradients and disruptions of progenitor patterning along thedorsal–ventral axis [78].Neural patterningIn developing sensory neurons, neurotrophin signalingpromotes axon outgrowth through clathrin-mediated endocytosis and signaling endosomes that are retrogradelytransported back to cell bodies to support neuronal survival[79, 80]. Zhou et al. showed that in addition to this “global”mechanism, neurotrophins also regulate sensory axonelongation and branching via a K63-mediated clathrinindependent TrkA endocytosis locally at the growth cone[81]. In cultured mouse embryonic dorsal root ganglionneurons, NGF stimulates K63 ubiquitination of Ulk1/2(Unc-51-like kinase 1/2) by TRAF6 and recruitment ofUlk1/2 to the TrkA receptor complex via interaction withp62, routing NGF-bound TrkA to non-clathrin-coatedSonic Hedgehog (Shh) signaling plays crucial roles invertebrate neural patterning, mediated by gradients of Ci/Glifamily transcription activators (GliA) and repressors (GliR).In a Shh-regulated manner, Glis are modified with degradative K11 or K48-polyUb chains by several multisubunitSCF E3 ligases (among others), which dictates the degradation and activator/repressor status [74–77]. Recent workindicates that Shh/Gli signaling gradient is also fine-tunedby non-proteolytic ubiquitination during ventral neural tubepatterning [78]. Specifically, RNF220, an E3 specificallyexpressed in the developing ventral neural tube, assemblesAxonal and dendritic outgrowth
Remodeling without destruction: non-proteolytic ubiquitin chains in neural function and brain disordersvesicles that attenuates NGF signaling and allows filopodiawithdrawal. This study highlights an important role forpresynaptic endosomal trafficking in axonal development(Fig. 3). Knocking down Ulk1/2 leads to impaired NGFendocytosis, excessive axon arborization, and severelystunted axon elongation. Mice lacking Ulk1/2 in the CNSshowed defects in axonal pathfinding and defasciculationaffecting the corpus callosum, anterior commissure, andcorticothalamic and thalamocortical axons [82]. However, itshould be noted that these loss-of-function experiments didnot directly address the role of K63 ubiquitination of Ulk1/2in these deficits.Another K63-dependent mechanism regulating neuriteoutgrowth is mediated by Muscleblind-Like Protein 1(MBNL1). MBNL1 is a multifunctional protein regulatingthe transition between differentiation and pluripotency andthe pathogenesis of myotonic dystrophy type 1 (DM1),which is associated with dendritic and synaptic transmissiondeficits at early stages [83]. MBNL1 undergoes K63 ubiquitination, which is required for its localization in thecytoplasm. Cytoplasmic, but not nuclear MBNL1 promotesaxon outgrowth and neurite differentiation in hippocampalneurons [84].Presynaptic differentiation and functionEndosomal trafficking at presynaptic terminals plays animportant role in synaptic vesicle (SV) cycles during presynaptic development and functions [85–87]. The ubiquitinbinding protein phospholipase A2 activating protein(PLAA) has been shown to act as a ubiquitin adaptorrequired for post-endocytic sorting of ubiquitinated presynaptic proteins from early endosomes to lysosomes fordegradation during SV recycling [88]. Mammalian PLAAspecifically recognizes K63-polyUb-modified SV components and targets them for ESCRT-dependent degradationvia MVBs. Mice deficient in PLAA show disrupted Purkinje cell migration and dendritic arborization, accumulatedK63-ubiquitinated proteins and presynaptic membraneproteins. This accumulation of K63-ubiquitinated proteinsis thought to be a result of impaired trafficking of theseproteins to ESCRT-dependent sorting and lysosomaldegradation. Mutant NMJs show striking presynapticswelling/sprouting, drastic loss of reserve pool SVs, andaberrant vesicle recycling during sustained activity. In bothhuman and mouse, hypomorphic mutations in PLAA causea lethal infantile neurodysfunction syndrome with epilepsy[88]. This study supports the importance of K63-relatedUBD-containing ubiquitin adaptors and endosomal trafficking in SV homeostasis and presynaptic differentiation(Fig. 3).In addition to its role in nuclear DNA repair in proliferating cells, RNF8 and associated K63-specific E2253Ubc13 regulate presynaptic differentiation in postmitoticneurons [89]. Knockdown or conditional knockout of RNF8or Ubc13 in mouse cerebellum granule neurons robustlyincreases the number of presynaptic boutons and parallelfiber-Purkinje cell synapses as well as functional synaptictransmission. Knockdown of the K48-synthesizing E2UbcH8, fails to increase the number of parallel fiber presynaptic boutons. Expression of an RNF8 mutant thatinteracts with Ubc13, but not UbcH8, rescues the number ofpresynaptic boutons. Cytoplasmic, but not nuclear RNF8exerts this presynaptic differentiation role. RNF8 interactswith the HECT domain protein HERC2/scaffold proteinNeuralized 4 (NEURL4) complex and knocking downeither protein mimics the inhibition of RNF8 on synapseformation. These results, though short of directly showinginvolvement of K63 chains, strongly suggest that RNF8/Ubc13-dependent K63-ubiquitin signaling inhibits synapseformation in vivo (Fig. 3). Detailed molecular details of thisK63-dependent presynaptic remodeling remain to bedetermined.The metabotropic glutamate receptor 7 (mGluR7), apresynaptic G Protein-coupled receptor that inhibits glutamate release especially during sustained and heightenedactivity [90], is K63 ubiquitinated following agonist stimulation in heterologous cells and cultured neurons [91].mGluR7 ubiquitination is mediated by Nedd4, which isrecruited to activated mGluR7 by the adaptor protein βarrestin, and leads to endosome–lysosome sorting anddegradation of the receptor. This process may play a role inthe regulation of presynaptic metaplastic long-termdepression (LTD)/long-term potentiation (LTP) at certainsynapses, e.g., mossy fiber-CA3 stratum lucidum interneuron (MF-SLIN) synapses [92] (Fig. 4; further discussedbelow).Postsynaptic function and dendritic spineremodelingIncreasing evidence indicates that K63 ubiquitinationoccurs at mammalian synapses. A mass-spec analysis of ratbrain ubiquitome shows that K63-polyUb species are thesecond most abundant ubiquitin linkage, behind K48 chains[16]. Mouse brain K63-polyUb levels increase duringpostnatal development and K63-polyUb clusters are abundant in dendritic spines in cultured rat hippocampal neurons[93]. K63-related ubiqu
Linear chains are also non-degradable by the proteasome and play important roles in NF-κB signaling [3, 19]. The remaining linkages are less prevalent and play degradative and/or non-degradative roles in more specialized cellular processes [4, 20]. K6 chains are implicated in DNA damage response (non degradative) and mitophagy (degradative).
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