Geobacter sulfurreducens are revisited. This bacterium has a diverse repertoire of chemosensory systems, which represents a striking example of a survival mechanism in challenging environments. Two G. sulfurreducens MCP—GSU0582 and GSU0935—are members of a new family of chemotaxis sensor proteins containing a periplasmic PAS-like sensor domain with a c-type heme. Interestingly, the cellular location of this domain opens new routes to the understanding of the redox potential sensing signaling transduction pathways. Environmental changes trigger the continuous adaptation of bacteria to ensure their survival. This is possible through a variety of signal transduction pathways involving chemoreceptors known as methyl-accepting chemotaxis proteins (MCP) that allow the microorganisms to redirect their mobility towards favorable environments. MCP are two-component regulatory  systems (TCS) formed by a sensor and a response regulator domain. These domains synchronize transient protein phosphorylation and dephosphorylation events to convert the stimuli into an appropriate cellular response. In this review, the variability of TCS domains and the most common signaling mechanisms are highlighted. This is followed by the description of the overall cellular topology, classification and mechanisms of MCP. Finally, the structural and functional properties of a new family of MCP found in  Escherichia coli (E. coli) genome encodes for 62 TCS proteins that are involved in the regulation of several processes, namely chemotaxis, osmoregulation, cellular metabolism and biological transport [Dictyostelium, TCS are also responsible for several metabolic processes, such as osmoregulation, growth and differentiation [Sensing environmental signals by chemoreceptors and the concomitant triggering of efficient adaptation responses, such as chemoattraction or chemorepellence, are key features of cell survival. Chemoreceptors constitute a highly diverse protein superfamily that differs in topology, cellular location, dimension, domain composition, function or even in the response mechanism. This review focuses on a particular group of chemoreceptors known as two-component systems (TCS). TCS are the most abundant multistep signaling pathways in Nature, and despite their crucial role in the cellular signaling strategies, the physiological signals that modulate their action are not precisely understood. TCS are found in organisms of all domains ; however, their abundance in each domain differs substantially . They arransport ,4. In Arransport ,6 and thransport ,8. In plntiation ,10. To dntiation ,12,13. TCS are important mediators of signal transduction characterized by a highly modular design, which has been adapted and integrated into a wide variety of cellular signaling events. These systems function as a basic stimulus-response coupling mechanism allowing organisms to sense and respond to distinct environmental changes and to perform a variety of functions, such as the alteration on the direction of the flagellar rotation or the regulation of the expression of a particular gene . The basic modus operandi encompasses phosphorylation events between a histidine kinase (HK) located at the sensor domain (SD) and the corresponding response regulator domain (RRD). SD is involved in the recognition of the chemical signal and the RRD triggers a particular cellular response. Both SD and RRD are composed by several subdomains .A typical HK sensor is constituted by two domains: a N-terminal input domain and a conserved C-terminal domain that interacts with the RRD. The N-terminal domain of the input domain is considerably variable. On the other hand, the RRD contains one conserved N-terminal receiver domain and one variable C-terminal output or effector domain ,15,16,17In most prokaryotic systems, the response is directly conducted by the RRD that functions as a transcription factor. However, in eukaryotic systems the TCS are placed at the start of the pathways and establish an interface with more conventional signaling strategies such as mitogen-activated protein (MAP) kinase and cyclic nucleotide cascades . Like moVibrio cholerae quorum sensing [Bacillus subtilis (B. subtilis) [Caulobacter crescentus (C. crescentus) [E. coli [B. subtilis [C. crescentus [Myxococcus xanthus [Parallel sensory kinases affect the domain with phosphotransferase activity within a more complex phosphorelay, such as the one observed for  sensing  or in thubtilis) . Many euscentus)  responsiscentus) , aerobic[E. coli ,23, the subtilis ,25 and tescentus ,27 and M xanthus ,29 have The kinase domains are part of a diverse range of SD, which allow the organisms to sense a wide range of signals, encompassing small molecules, light, pH, osmotic changes, nutrient levels, quorum-sensing proteins, redox potential and electrochemical gradients. SD are highly variable in sequence, particularly those flanked by cytoplasmic transmembrane (TM) domains . In thisDifferent membrane topologies were observed for SD and are divided into three main groups. The first one encompasses the cytoplasmic SD. The second group includes SD with multiple membrane-spanning helices but without an apparent extracellular domain. The stimuli detected by these HK are most likely associated to alterations of the membrane components and ion gradients. The last and largest group of SD is represented by the classical HK with extracytoplasmic domains placed between two TM helices. These SD include several families that transduce the extracellular stimulus across the membrane to regulate the activity of kinases/phosphatases. In these cases, multiple topologies can be combined for a concerted response to environmental changes.Multiple cytoplasmic SD have a so-called PAS domain  and/or a GAF domain .  bacteriophytochrome photosensory core domain (PaBphP-PCD) and revealed a tripartite photosensory core domain (PCD) containing PAS, GAF and phytochrome (PHY) domains [Dienococcus radiodurans DrBphP-CBD [Rhodopseudomonas palustris [Synechocystis 6803 Cph1 [Phytochromes from fungi, bacteria and plants constitute the third family of cytoplasmic SD. This family includes soluble HK photoreceptors. Structural data were obtained for the  domains ,41. LikeBphP-CBD , the RpBalustris  and PCD 803 Cph1 .HK receptors containing several TM segments frequently miss an obvious extracellular domain. In these cases, the sensing region is found within the TM helices. Complementary biochemical and biophysical studies have confirmed this hypothesis in the case of the DesK temperature sensor ,46. In tE. coli, (ii) β-sheet fold like the P. aeruginosa RetS periplasmic detector domain and (iii) a mixed α/β fold named PAS-like/PDC [Three classes of extracellular SD have been established according to their sequence similarity and fold ,52: (i) like/PDC  and, morlike/PDC . The amilike/PDC ,55.The new nomenclature proposed for extracytoplasmatic PAS-like or PDC domains—Cache—is based on an extensive bioinformatic study carried out by Upadhyay and co-workers  in whichAdditions to the structural collection of sCache sensors are provided by the oligosaccharide sensor AbfS  and the B. subtilis that regulate the YycG sensor kinase [Bacillus anthracis that control the sporulation [The activities of some sensors are regulated by auxiliary proteins. In these cases, the binding of such proteins is in fact the primary ligand-binding event. The structures of some proteins that are responsible for the control of their respective sensor kinases have been determined: LuxP that regulates the LuxQ activity; YycH and YycI from r kinase ,58 and trulation .2+ and Fe3+. The iron atom can be penta- or hexacoordinated and, depending upon the axial ligands, it can be high-spin (HS) or low-spin (LS). The interconversion between different spin states, as well as between different oxidation states can be explored by the SD to trigger an adequate cellular response. Heme proteins are one of the most versatile group of proteins in Nature. In addition to their well-known role in electron transfer, oxygen transport or reduction of peroxides, they can also be involved in chemotaxis and regulation of gene expression . The hemThe currently known heme-based sensors are grouped into six different families according to their heme-binding domains: (i) heme-nitric oxide binding domain (H-NOX), (ii) globin-coupled sensor fold, (iii) heme binding PAS fold, (iv) GAF domain (v) bacterial CO oxidation transcriptional activator (CooA) homologues, and (vi) the sensor containing heme instead of cobalamin (SCHIC) domain of anoxygenic phototrophic proteobacteria . All theRhizobium meliloti and EcDos in E. coli), GAF domains (Mycobacterium tuberculosis (M. tuberculosis), DosS and DosT) and in globin domains (B. subtilis HemAT). The PAS domains usually contain heme cofactors, while GAF domains frequently have cyclic nucleotides.The structural variability of the heme-containing sensors correlates with their diverse role in living systems. Heme groups have been reported in PAS domains  of M. tuberculosis DosS [FixL is a heme-based oxygen sensor which controls the expression of specific proteins under microaerobic or anaerobic conditions ,67,68 anme group . The sam[E. coli . This se[E. coli ,72. A co coli [1 , suggestsis DosS  and DosTsis DosS , which db-type hemes, few TCS containing c-type hemes have been identified to date. One example is the multidomain protein MA4561 from Methanosarcina acetivorans [In contrast with tivorans , which iThermosynechococcus elongatus protein Tll0287 has a PAS-like domain with a c-type heme and was identified as a TCS involved in signal transduction in response to environmental factors related to the sulfide metabolism [The tabolism .c-type hemes were identified in chemoreceptor proteins from Desulfovibrio vulgaris (D. vulgaris) Hildenborough (DcrA) [G. sulfurreducens  [c-type (DcrAN) responsible for sensing redox potential and oxygen variations. It is the first example of a heme-based sensor protein containing a c-type heme reported in the literature. This protein has a cytoplasmic PAS domain without the heme cofactor. Thus, it was proposed that the cytoplasmic PAS domain of DcrA may be involved in the output response to the signal sensed by the heme c in the periplasmic space.PAS domains containing h (DcrA)  and G. sGSU0935) ,79. DcrAIn prokaryotes, RRD are usually the last components of the signaling pathways and are directly involved in the effecting responses. On the other hand, in eukaryotes, RRD act as intermediates and establish the necessary interface with proteins that regulate kinase cascades or the production of nucleotide-based second messengers . Only a It has been suggested that signal transduction via HK domains mostly requires the formation of a dimer. However, several SD transmit the signal in their monomeric form  ,92,93,94Structural data obtained in recent years for several extracytoplasmic and cytoplasmic SD permitted to establish different classes of proteins sharing similar folds. Vibrio harveyi. In this case, the ligand binds to a periplasmic protein (LuxP), which then drives the formation of an asymmetrical dimer that transmits the signal to cytoplasmic domains and inhibits the kinase activity [E. coli and Salmonella enterica or CitA from K. pneumoniae.The structural information obtained for Tar and systems like EnvZ and NarX, suggested the existence of a piston-like movement that propagates the signaling via the TM helices into the cytoplasm ,99. Howeactivity . In contThe physiological relevance of the proteins’ quaternary structure remains puzzling since isolated Cache domains have smaller propensity for dimerization and their plasticity creates distinct orientations of the dimers’ subunits in the crystals. On the other hand, such heterogeneity might reveal distinct signaling stages and hence contain valuable information.Some Cache structures suggest a common feature of the N- and C-terminal regions for these periplasmic SD by placing the TM helices at locations consistent with a four-helix bundle matching those of Tar and NarX ,100,101.E. coli and Salmonella enterica serovar Typhimurium for the regulation of their flagellar-based motility [Chemotaxis is intimately linked to bacterial growth and survival and is the key for the movement towards environments containing either higher concentrations of beneficial compounds or lower concentrations of toxic chemicals. The bacterial chemotaxis machinery was firstly identified in motility ,109,110.motility . This suE. coli B275 strain required L-methionine to grow and perform chemotaxis. Using radioactive methionine, the authors showed that the methyl group is incorporated into a protein located in the cytoplasmic membrane—the methyl-accepting chemotaxis protein [Nearly 50 years ago, Julius Adler and Margaret Dahl  observed protein . As a re protein ,115,116.Archaea. The best investigated is the Tar chemoreceptor from E. coli, which has four MCP  and one MCP-like protein (Aer). Roger Alexander and Igor Zhulin [Bacteria and Archaea. Barbara A. Methé and co-workers [G. sulfurreducens genome, 34 MCP genes and six major che gene clusters. MCP are the predominant chemoreceptors in bacteria and have been found in many r Zhulin  identifi-workers  discoverGeobacter metallireducens , G. sulfurreducens and Geobacter uraniireducens (G. uraniireducens) genomes, 90% have two TM helices. The majority (80%) contain periplasmic domains with about 150-200 amino acids and are comparable to the periplasmic domains of E. coli MCP Tsr [G. sulfurreducens (GSU0935 and GSU0582) were established and revealed PAS-like domains containing c-type heme groups [Until 2003, the only structure of a periplasmic ligand-binding domain available was the one of the chemoreceptor Tar that encompasses a four-helix bundle motif . From th MCP Tsr . While te groups    Figure . Later, Wuichet and co-workers  have subThe members of class I have a periplasmic ligand-binding domain placed between two TM helices and a cytoplasmic component containing the methylation sites and a conserved domain that regulates the HK activity ,123,124.Class II receptors harbor a single hydrophobic patch between a putative SD, a cytoplasmic HAMP region and a cytoplasmic signaling module. The class II receptors  are found solely in Bacteria and are much less abundant (3%) compared to the members of the other classes . Synechocystis is a typical representative of class III [Transducers from class III have a distinct number of membrane-spanning helices with the SD placed either within the membrane (subclass IIIm) or in the cytoplasm (subclass IIIc). In both cases, the SD is followed by the cytoplasmic HAMP region and the signaling domain. The subclass IIIm lacks an extracellular domain and the stimuli is most likely membrane-associated. The photoreceptor from lass III . The patThe MCP from class IV are cytoplasmic containing (subclass IVa) or not (subclass IVb) an identifiable SD. These MCP lack TM helices as well as, in most cases, the HAMP domains. The mechanism of ligand recognition in subclass IVb is still under debate.There are some hybrid systems identified for classes II and III, which combine periplasmic and cytoplasmic SD . MCP sequence comparison indicates that the SD tend to be highly variable, whereas the cytoplasmic signaling domains that interact with the downstream regulatory proteins are highly conserved. The middle components of the chemotaxis signaling pathway, placed between the MCP receptors and the flagella are the Che proteins . che genes are widespread in Bacteria and Archaea. The genes respond to the MCP’s interaction with an attractant or repellent. CheA, CheW, CheY and CheZ transmit the flagellar response. Subsequently, CheB and CheR conduct the adaptation to the stimulus by methylating or demethylating the MCP. Receptor MCP are coupled to an adaptor protein CheW (or CheV), to a CheA HK, and two RRD (CheB and CheY) that compete for binding to CheA. CheY is a specialized single-domain flagellar motor-binding protein that regulates the response, whereas CheB has two domains, one responsible with methylesterase activity and controls the adaptation of the MCP and a second one that functions as a response regulator [E. coli chemotaxis, an auxiliary protein (the phosphatase CheZ) oligomerizes with the CheY∼P and increases the spontaneous dephosphorylation rate of CheY∼P for rapid signal termination [The egulator ,128. Theegulator ,130. Theegulator ,132. Chemination .The cells’ capability to detect and respond to small concentration changes of attractant and repellents is intimately associated to the diversity of their chemoreceptors. The levels of CheY∼P increase with the decrease of the attractant. Consequently, the flagellar motor switches to clockwise rotation and alter the cell tumbling and direction. On the other hand, the demethylated MCP, as a response to the higher levels of CheB∼P, reduces the autophosphorylation rate of CheA and therefore the switch rate of the flagellar motor rotation reverts to the pre-stimulus state. The system is then adapted and ready to sense any subsequent variation on the signal molecule(s). On the other hand, an increase in the concentration of the attractant will inhibit the autophosphorylation of CheA and decrease the frequency of the motor switching, in response to lower levels of CheY∼P. This event triggers the bacterium to swim in this new direction. Then, CheB∼P decreases, which favors the methylation of MCP by the methyltransferase CheR. This brings about the CheA autophosphorylation to the pre-stimulus state and allows the bacterium to restore its typical direction changing .Geobacter are facultative anaerobes Gram-negative δ-Proteobacteria with extraordinary respiratory diversity. Geobacter are abundant in soils and sedimentary habitats capable of completely oxidizing organic compounds to CO2 [Geobacter are insoluble under environmental conditions. Due to their ability to transfer extracellular electrons, these bacteria are considered as attractive targets for several practical applications for bioremediation of radioactive/toxic metals in contaminated subsurface environments and for bioenergy generation by converting organic compounds into electricity in microbial fuel cells [Geobacter cells proliferate these organisms contain a high number of MCP chemoreceptors. The ability to respond efficiently in extreme environments, such as the saturation of a specific electron acceptor, the replacement by different electron donors or even changes in the surroundings redox potential, is an essential feature of Geobacter bacteria, which promote well-organized metabolic adjustments.s to CO2 . Many ofel cells ,136,137.Geobacter genomes revealed an abundance of che gene homologs and multiple chemotaxis systems [Recent analysis of sequenced  systems  , with an amino acid signature of CX2–4CH. Eight of these proteins are annotated as two-component system proteins , two as chemotaxis signal transducer proteins (GSU0582 and GSU0935), one as cytochrome c family protein (GSU0591) and one as HAMP/GAF/HD-GYP protein (GSU2622) [G. sulfurreducens’ signal transduction proteins, nine have homologs in G. metallireducens [equenced  and for equenced . The anaGSU2622) ,140. Amoreducens .c, connected via two TM to the cytoplasmic domain, which consists of a HAMP domain followed by a MCP domain. The proposed action mechanism involves the sensing of the external stimulus by the periplasmic SD that then transmits the signal via the TM helices, activates its cytoplasmic transducer domains and triggers the response to the original extracellular signal [The sequence alignment of the MCP heme sensors GSU0582 and GSU0935 indicates that they have a periplasmic domain containing a heme r signal   of the two sensor domains, the crystal structures  showed t143 and by a water molecule. On the other hand, in GSU0935 dimer, one of the hemes is HS with axial ligands His144 and a water molecule while the other heme is LS—hexacoordinated with His144-Met60 ligation. Complementary spectroscopic experiments, including electron paramagnetic resonance (EPR), nuclear magnetic resonance (NMR) and optical absorption spectroscopy showed that the hemes are HS and LS in the oxidized and reduced forms, respectively. It was also shown that the interconversion between the HS and LS forms is driven by the replacement of the heme distal axial ligand (see below) [The swapped region encompasses two α-helices at the N-terminal and in the dimer the hemes are placed between the α-helices of one monomer and the β-strands of the other . In the e below) .K. pneumoniae  and LS (S = 0) in the reduced form. This spin change is probably caused by the binding of Met60. It was also showed that both sensors can bind carbon monoxide (CO) in the reduced form but not in the oxidized form. On the other hand, nitric oxide (NO) binds in both forms. The binding of CO and NO to the reduced heme replaces the axial Met60 and the binding/dissociation of the two diatomic molecules is fully reversible. The functional properties of oscopies . These sThe data obtained from resonance Raman experiments, molecular dynamics calculations and binding studies revealed several common features for both sensors: (i) co-existence of two spin populations in the oxidized state; (ii) reduction of the heme favors the LS population; (iii) CO binding forms a hexacoordinated (6c) LS-CO species; (iv) NO binding forms yields a pentacoordinated (5c) HS-NO and 6cLS-NO species in both redox states. The data also suggested that in the reduced form the sensors are unable to discriminate between the two diatomic molecules, whereas in the oxidized GSU0582 has a much higher affinity for NO . OverallDespite the structural similarity of GSU0582 and GSU0935, the results obtained from the potentiometric redox titrations indicate that these sensors are functionally distinct. Unlike GSU0582, the GSU0935 sensor domain displays significant variations in the redox potential: −156 mV for GSU0582 and −251 mV for GSU0935, respectively. The differences observed are most likely caused by the nature of the amino acid residues in the vicinity of the heme groups . The facTCS are abundant multistep signaling pathways that respond to a wide range of stimulus. Several components of the TCS have been structurally and/or functionally characterized. For a large majority of TCS, how physiological signals modulate their action is not precisely understood; however, such studies have provided crucial information regarding the modus operandi of TCS. Most HK and RR share the basic structural and functional properties. However, TCS proteins usually exhibit specificity for intermolecular interactions (dimerization). The modular HK and RR architectures permit significant variations in the arrangement of their domains. The signal-linked interconversion changes usually involve the phosphorylation of residues in specific domains, favoring distinct intra- or intermolecular protein–protein interactions that activate or inhibit a particular cellular response. The arrangement of the domains, and hence the regulatory mechanisms, could differ amongst members of the same subfamily. Thus, a proper prediction of such arrangements and precise mechanism still requires further investigation.The efforts made up until now to characterize systems such those involved in bacterial chemotaxis have provided a broad understanding of how extracellular signals are transduced and allow cells to trigger proper responses. Contrarily to the signaling domains, which are conserved and always located in the cytoplasmic, the sensory domains present significant variations in their structure and membrane topology. The PAS and PAS-like domains are ubiquitous and sense directly or indirectly a large variety of internal or external stimuli. In the future, it would be not surprising if new ligands are discovered and more PAS and PAS-like domains identified and characterized. Signaling systems involved in bacterial chemotaxis are highly specialized TCS that during the evolution have adapted the receptor domains in a way that provided the bacteria a high plasticity for input, output and regulation. Organisms that inhabit challenging habitats control their motility and perhaps other yet unveiled processes, by using these specialized systems. The increasing number of chemosensory systems characterized opens new avenues to the clarification of the signal amplification mechanisms . DespiteG. sulfurreducens. These proteins are the first members of a newly described bacterial class of c-type heme-based sensors. Following the environmental fluctuations in redox potential, conformation changes observed at the heme moiety of these two sensors match those occurring at the global protein structure, suggesting that signaling at the heme group induces conformational alterations that are transmitted to the other protein domains to complete the signal transduction cycle. The characterization of these two chemotaxis sensors provided additional understanding of bacterial signal transduction mechanisms showing that the sensors operate over large and distinct redox potential ranges by linking changes in their heme spin state/coordination to structural alterations that are transmitted to the cytoplasmatic regulatory domains. One may speculate that the same mechanism may be shared by other c-type heme-based sensors in G. sulfurreducens and other bacteria. The distinct midpoint reduction potential values of each sensor domain suggest that they are functionally relevant in different redox potential ranges with GSU0935 and GSU0582 operating, respectively at lower and higher redox potentials. These outcomes provided for the first time a justification for the co-existence of two similar MCP in G. sulfurreducens and how they are designed to allow the bacteria to activate an adequate cellular response in highly variable subsurface habitats. Heme proteins are involved in many different types of biological processes, including chemotaxis. Examples of proteins involved in this process are the GSU0582 and GSU0935 sensors from


DNA polymerase eta (Polη) is a unique translesion DNA synthesis (TLS) enzyme required for the error-free bypass of ultraviolet ray (UV)-induced cyclobutane pyrimidine dimers in DNA. Therefore, its deficiency confers cellular sensitivity to UV radiation and an increased rate of UV-induced mutagenesis. Polη possesses a ubiquitin-binding zinc finger (ubz) domain and a PCNA-interacting-protein (pip) motif in the carboxy-terminal region. The role of the Polη pip motif in PCNA interaction required for DNA polymerase recruitment to the stalled replication fork has been demonstrated in earlier studies; however, the function of the ubz domain remains divisive. As per the current notion, the ubz domain of Polη binds to the ubiquitin moiety of the ubiquitinated PCNA, but such interaction is found to be nonessential for Polη's function. In this study, through amino acid sequence alignments, we identify three classes of Polη among different species based on the presence or absence of pip motif or ubz domain and using comprehensive mutational analyses, we show that the ubz domain of Polη, which intrinsically lacks the pip motif directly binds to the interdomain connecting loop (IDCL) of PCNA and regulates Polη's TLS activity. We further propose two distinct modes of PCNA interaction mediated either by pip motif or ubz domain in various Polη homologs. When the pip motif or ubz domain of a given Polη binds to the IDCL of PCNA, such interaction becomes essential, whereas the binding of ubz domain to PCNA through ubiquitin is dispensable for Polη's function. Y-family DNA polymerases (Pols) are a group of nonessential enzymes that play an imperative role during translesion DNA synthesis (TLS) , 2. TheySaccharomyces cerevisiae and humans, genetic and biochemical studies have indicated that PCNA plays a pivotal role in the pol exchange process (PCNA interacting protein (pip) motif(s) present mostly in the noncatalytic region of the pols  abrogates its interaction with PCNA  at the C-terminal domain . As PCNA gets monoubiquitinated via Rad6-Rad18 ubiquitination conjugating system during TLS . Since similar to CaPolη, other 22 Polηs of category III also lack the pip motif (B). The purified wild-type CaPolη protein  was mixed with CaPCNA in a 1:1 M ratio and allowed to resolve in analytical size-exclusion chromatography. For comparison, CaPCNA alone was also passed through the column, and its elution profile was recorded. As reported earlier also, CaPCNA (∼90 kDa) eluted as a trimer with an elution volume of ∼1.7 ml . In addition, a unique proline-rich sequence located in between Seq-4 and ubz domain of CaPolη is observed that might provide additional structural flexibility to the C-terminal portion to be engaged in protein–protein interaction. To identify a region of CaPolη involved in PCNA interaction, we generated a deletion construct by inserting a stop codon at amino acid position 513 (K513) of CaPolη. The truncated CaPolη spanning residues 1 to 512 encodes for the whole catalytic domain up to the PAD (D), and similarly truncated Polηs both from S. cerevisiae and human are known to retain DNA polymerase activity as good as the full-length enzymes  function in the error-free and error-prone branches of TLS of UV lesions, respectively; therefore, double deletant S. cerevisiae (rad30Δrev3Δ) strain becomes hypersensitive to UV radiation than the individual null strain , and the sensitivity was similar to that of the vector control. Thus, the catalytic domain consisting of the first 512 amino acids of CaPolη is insufficient to carry out lesion bypass in the cell as well as inefficient in PCNA binding. Moreover, a similar complementation assay with CaPolη F485A and L486A mutations that supported the growth of cells upon UV exposure ruled out the involvement of Seq-2 of PAD in PCNA interaction . These results implicated the requirement of the C-terminal domain region spanning residues 513 to 640 aa of CaPolη in TLS and possibly in PCNA interaction.The direct interaction of CaPolη with CaPCNA implicated the involvement of yet unidentified pip motif or domain of CaPolη in PCNA binding. Four putative noncanonical pip sequences: Seq-1 (390SFVISNLF397), Seq-2 (479CVLIREFL486), Seq-3 (521SLTISKFV528), and Seq-4(547EEHIAKLF554) in and around the PAD of CaPolη may bind to PCNA C. In add the PAD D, and si enzymes . The wiler alone . It sugger alone , 25, 26,l strain . As depiA). Similar to CaPolη 1 to 512 aa, Polη 1 to 601 aa also conferred significant growth retardation at 16 to 32 J/m2 of UV irradiation in both rad30Δ and rad30Δrev3Δ S. cerevisiae strains implying that both Seq-3 and Seq-4 are not essential for CaPolη's TLS activity in the cell and are unlikely to be involved in PCNA interaction (B). Moreover, it suggested the critical role of the C-terminal 39 amino acids comprising the ubz domain of CaPolη in TLS activity. To ascertain the function of ubz domain, two D626A and H624A, H628A of CaPolη mutants were generated and their ability to suppress UV sensitivity of Polη-deficient S. cerevisiae strains was examined. Both the CaPolη mutants did not confer any cellular protection to UV radiation, and growth was inhibited drastically (B). The complementation analysis was reconfirmed by estimating the cell survivability upon UV exposure; where about 85 to 95% of cells expressing wild-type Polη conferred resistance, only ∼10 to 35% rad30Δ cells harboring CaPolη mutants survived after 32 J/m2 of UV treatment . Similarly, UV-induced mutation rate was estimated by counting the number of canavanine-resistant colonies of various S. cerevisiae strains harboring CaPolη constructs. The rad30Δ strain or strains harboring truncated or ubz mutants of CaPolη were highly mutagenic as more number colonies (∼4000 per 107 cells) grew as canavanine-resistant, as opposed to the strains expressing wild-type Polη . This result suggested that the ubz domain of CaPolη plays an essential role in the error-free bypass of UV-induced lesions and possibly in PCNA interaction. Subsequently, ITC and GST-pull-down assays were carried out to determine the role of ubz in PCNA binding. About 10 μM of each mutant CaPolη 1 to 601 aa, D626A, and H624A, H628A proteins were placed in the calorimetric cell, and the change in the heat was monitored after each CaPCNA injection . Our ITC assays revealed no significant net heat change when PCNA was titrated against the mutants of Polη suggesting no detectable interaction between the proteins. Similarly, the GST-pull-down assay authenticated the ITC results where wild-type GST-Polη pulled down PCNA from the mixture, whereas the mutant Polη proteins failed to pull down PCNA . From these observations, we concluded that the ubz domain of CaPolη directly binds to PCNA, and this interaction is essential for its in vivo activity.The above results indicated that the C-terminal portion of CaPolη containing two putative pip sequences: Seq-3 and Seq-4, and one ubz domain could be involved in PCNA interaction. To map the precise location of PCNA binding, another deletant CaPolη 1 to 601 aa was constructed that lacks the last 39 residues encompassing the ubz domain but retains Seq-3 and Seq-4 A. Similaeraction B. Moreovstically B. The coreatment C, i. SimA). While human and C. albicans Polηs possess a canonical C2H2-Zn2+-binding domain, ScPolη ubz is noncanonical as the second cysteine residue is replaced by glutamine. Although there is hardly any similarity in the primary sequences among these ubz domains except for the positions of cysteines and histidines that coordinate the metal cofactor, they adopt a classical superimposable C2H2 zinc finger structure characterized by a β-fold comprised of two short antiparallel strands and a carboxyl-terminal α-helix. The two cysteines are located on the fingertip made by the two β-strands, and the two histidines are located on the α-helix. The α-helix shows the maximum sequence identity and is found to be involved in interaction with ubiquitin  and its corresponding mutant peptide (607-KTKKCSRCKLSVDDPVEANDYAIAMDLSN-635) were synthesized, and their interaction with CaPCNA was validated by ITC analysis. While PCNA titration against the wild-type ubz peptide taken in the calorimetric cell resulted in the release of heat, it did not show any sign of binding with the mutant peptide as no net energy change was detected . The kinetic parameters upon the binding of wild-type ubz peptide with PCNA were determined as ΔH = −80 kcal/mol, ΔG = −6.42 kcal/mol, and KD = 19.8 μM , but a mixture of ScPCNA and ubz peptide excluded from binding to ScPolη . To further strengthen our finding, a GST pull-down assay was carried out to pull down PCNA from an equimolar mixture of GST-CaPolη with wild-type or mutants of CaPCNA proteins. While GST-CaPolη was able to pull down most of the wild-type PCNA and PCNA-90 proteins from the solution , it failed to bind PCNA-79 (compare lane 4 with 6) (A). Both the assays suggested that the hydrophobic cavity formed by the IDCL of PCNA is the interaction site of CaPolη, mediated by its ubz domain.The PCNA interaction motif of the ligand occupies the hydrophobic pocket formed by the IDCL that connects the two globular domains of a PCNA protomer , 34. MosCNA ring D. To fur with 6) A. Both tA and B; F627A, F628A mutations in the pip box of ScPolη abrogated its TLS function and displayed equal UV sensitivity with rad30Δ strain but H568A, H572A mutations in ubz of ScPolη did not affect survival and restored wild-type level of UV sensitivity to the rad30Δ strain (compare sectors i and iii with ii and iv). Thus, while the pip motif of ScPolη plays a critical role in TLS, the ubz of ScPolη is nonessential, and this result further authenticates our earlier reports . Interestingly, the carboxyl-terminal fusion of ScRad30-pip sequence to these mutants rescued the UV sensitive phenotype in the rad30Δ strain. At 16 to 32 J/m2 of UV, a significant level of growth was observed for rad30Δ strain expressing CaPolη D625A∼ScRad30pip or CaPolη H624A, H628A∼ScRad30pip or CaPolη ubzΔ∼ScRad30pip chimeras than the vector control alone suggesting that presence of ScRad30 pip motif alone is sufficient to suppress UV susceptibility, and ubz domain is dispensable (compare sectors xi–xiii with i). Interestingly, a similar fusion of ScRad30-pip to wild-type CaPolη conferred slightly better resistance to UV irradiation than the rad30Δ strain expressing CaRad30 (compare sectors x with ix). Although the presence of ubz of CaPolη is essential for its TLS function, the fusion of this domain alone to the catalytic domain of ScPolη (ScPolη CTDΔ∼CaRad30 ubz) partially rescued the UV-sensitive phenotype of rad30 deletion strain . At a higher dose of UV radiation (32 J/m2), ubz fused ScPolη failed to carry out efficient TLS. We obtained almost similar results when we tested these hybrid Polηs in rad30Δrev3Δ, yet another Polη-deficient S. cerevisiae strain (B). Overall, these results suggest that a ubz domain can substitute for a pip motif in CaPolη for TLS activity but not the other way in ScPolη. Probably, not so conserved flanking sequences in Polηs might be playing a role in deciding whether ubz can function as a pip motif or not.Since ubz of CaPolη functionally mimics the pip motif of ScPolη, to understand the species-specific roles of these two critical motifs during TLS, an array of hybrid Polη constructs were generated and analyzed . The pip reports , 17. Howe strain B. OveralC. albicans are due to its translesion DNA synthesis activity, while its TLS-independent functions play a pivotal role in serum-induced morphogenesis and amphotericin B resistance. C. albicans cells harboring CaPolη with catalytically inactive mutations  exhibited similar phenotypes as the rad30ΔΔ strain . Only the knockout strain expressing wild-type CaPolη survived to a higher dosage of UV irradiation (16–32 J/m2). Thus, like in S. cerevisiae, to carry out translesion DNA synthesis even in C. albicans, CaPolη requires both the catalytic and the ubz domains. Further these strains were exposed to sublethal dose of UV radiation and allowed to repair the damage by growing them on fresh media. Since TLS activity of Polη is required to bypass DNA lesions to prevent replication fork collapse and accumulation of DNA breaks, any strains failing to conduct TLS will accumulate fragmented smaller sized chromosomal DNA, while the ability of Polη to bypass UV-induced lesions will lead to the accumulation of longer chromosomal DNA. Alkali agarose gel electrophoresis of total genomic DNA isolated at various time points of recovery revealed that an equal amount of genomic DNA degradation was found in each cell type prior to recovery . The longer the duration of the recovery period, the more was the accumulation of larger DNA fragments, and a lesser amount of smaller chromosomal DNA fragments were accumulated in the cells expressing wild-type than expressing ubz domain deletion or its site-directed CaPolη mutants . This result suggested that C. albicans expressing CaPolη was proficient in carrying out UV-inflicted DNA lesion bypass than the cells possessing Polη ubz mutants, and this could be because of the inability of the CaPolη ubz mutants to interact with CaPCNA for efficient lesion bypass. To further strengthen our result, the requirement of ubz domain of CaPolη in maintaining genomic stability was determined by estimating the loss of functionality of URA3 gene upon 5-fluoroorotic acid (5-FOA) treatment with and without UV irradiation. First, we generated a heterozygous URA3/ura3-in Polη-deficient C. albicans strain, and the loss of heterozygosity (LOH) was estimated through a 5-FOA resistance assay . We found that without UV irradiation, the rate of LOH in various rad30ΔΔ C. albicans strains remained unaltered (5–8 per 5 × 105 cells), even it was the same in wild-type Polη expressing cells when exposed to UV. However, the number of FOA-resistant colonies increased by ∼three-fold in the cells expressing ubz deletion and H624A, H628A mutants of Polη (16–25 per 5 × 105 cells) upon UV exposure.In our earlier studies, we reported that the roles of CaPolη in genome stability, genotoxins-induced filamentation and azole drug tolerance by Δ strain , 27. To ty assay . The C.  control A. Only tC. albicans pathogenesis. Certain gene knockout strains locked in either round or hyphal structures are avirulent in developing systemic fungal infections in animal models. Filamentation in C. albicans is induced by elevated temperatures, serum, spider media, DNA damaging agents, and other specific nutrients . The auxotrophic BWP17 strain of C. albicans does not undergo filamentation due to the absence of functional URA3 gene; however, an ectopic expression of URA3 by its integration into the genome results in germ tube formation induced by serum  for 1 h before checking their cellular morphology. Except for the strain expressing wild-type Polη, where 97% of C. albicans cells developed germ tubes, only ∼50% of cells from other ubz defective strains developed germ tubes; that also with substantially reduced lengths (E and F). Consequently, more round cells were counted in C. albicans cells without the functional ubz domain. These results suggest that CaPolη without ubz domain exacerbates morphological defects induced upon serum addition. Taking it all together, we conclude that the ubz domain of CaPolη plays a pivotal role in TLS-mediated genome stability and morphogenesis in C. albicans.Morphogenesis plays an important role in utrients . C. albiormation , 27. To ormation , D–F. Thby serum . First,  lengths , E and FS. cerevisiae and human Polη gain access to PCNA via a pip motif, CaPolη that intrinsically lacks a pip motif interacts with PCNA through its ubz domain for the TLS activity. Thus, for S. cerevisiae and human Polη where a pip motif is present, ubz becomes dispensable; and for C. albicans Polη where pip is naturally absent, the role of ubz becomes imperative. To understand the evolutionarily conserved role of the ubz domain of Polη, we selected a few other members of Polη from category III that lack canonical PCNA interaction motif at the carboxyl-terminal tail. We expressed wild-type Polηs from S. pombe, Neurospora crassa, and Aspergillus nidulans in rad30Δ S. cerevisiae strain for complementation analysis (A). Unlike CaPolη, Nc- and An-Polη failed to complement the function of ScPolη, whereas SpPolη (872 aa) partially complemented and supported the growth even at a higher dosage of UV. However, the deletion of the ubz domain of SpPolη (1–682 aa) completely abolished resistance to UV; thus, similar to CaPolη, the ubz domain of SpPolη is essential for its TLS activity (compare sectors v and vi with i). To strengthen the spot assay, we estimated the colony formation units when cells expressing either SpPolη or SpPolη ubzΔ were exposed to UV radiation. The S. cerevisiae cells expressing wild-type SpPolη formed more colonies upon treatment with UV than the cells expressing ubz deletion mutant of SpPolη, which is similar to a Polη-deficient strain . Although we could not find complementation by Nc- and An-Polηs in rad30Δ S. cerevisiae strain, the results obtained from ubz mutational analyses of Ca- and Sp-Polηs clearly supported the indispensable conserved role of ubz of category III Polη in TLS. Considering earlier studies and this study, we propose two modes of Polη recruitment to PCNA, mediated either by pip box or ubz domain in three categories of Polηs (C and D). For category I and II Polηs, pip motif plays an essential role, and ubz/ubm, if present becomes dispensable, whereas in category III Polηs, as they lack pip motif intrinsically, ubz domain interaction with the PCNA becomes critical. While in category I, ubz may interact indirectly with PCNA mediated by the ubiquitin moiety, in category III Polηs, ubz directly binds to the IDCl of PCNA similar to the pip motif of category I and II Polηs.From the above results and our earlier observations, it is evident that while analysis A. UnlikeTo gain access to the replication fork, whether, during replication or translesion DNA synthesis, DNA polymerase needs to interact with PCNA, a step that is considered to be essential , 3. TherS. pombe partially complemented ScPolη, analyses of its ubz mutant further supported the significance of this domain in TLS function. Thus, we proposed that where pip motif is naturally absent, the ubz domain of Polηs could play a critical role in PCNA interaction. In contrast to category III Polηs, the representative studies on Polηs from categories I and II suggested the indispensable nature of the pip motif in PCNA interaction and for their TLS activities, thereby the existence of two distinct modes of PCNA interactions among Polηs mediated either by pip or ubz domains is possible . Moreover, Polηs from protists such as Trypanosoma, Leishmania, Caenorhabditis elegans of category II also lack ubz completely, but they have pip-like motifs at the C-terminal domain. Since the mutations in the C2H2 motif of ScPolη and HsPolη exhibited either no or weak phenotypes when compared with their pip mutants and ubz is absent in category II Polηs, the role of ubz becomes dispensable for the TLS function in category I and II Polηs . Thus, even in S. cerevisiae Polη, the ubz domain can bind to PCNA provided the pip is completely absent in the C-terminal domain. Unlike the pip motif that forms a flexible 310 helix and gets stabilized into the hydrophobic pocket of the IDCL in PCNA trimer, ubz is structurally more organized, consisting of two antiparallel β-strands and an α helix . Ubz and its mutant peptides were synthesized with 98% purity and procured from China peptides Co Ltd. The wild-type strains of N. crassa Rad30 contains a single intron within the orf, using its cDNA as a template and NAP438 (5′-CCG GGG ATC CAC ATA TGT CGT CAC CGC CC-3′) and NAP347 (5′-CCG GGG ATC CTC AAC CGA ACT TGA GTT TGC -3′) primers NcPolη orf was amplified. To generate truncated Rad30 orfs, NAP03 and NAP08 (5′-CCG GGG TAC CGG ATC CTC ATT ACA AAA GGG TAA ATC TGT C-3′) for CaRad30 (1–512); and NAP343 and NAP345 (5′- CCG GGG ATC CTC ACT TTT TCC AAT TTG GTG TCA GAT TTC C) for SpRad30 (1–628) primers were used for amplification. In the case of CaRad30 (1–601) truncation, an SMURFT linker was ligated into the unique XbaI site of the CaRad30 orf construct. Similarly, by using NAP03 and a set of reverse primers containing either D626A mutation in NAP333 (rp 5′- CCG GGG ATC CTT AAT GAT TAT TTA GTT TGT TTG ATA AGT CCA TCG CTA TGT GGT AGG CGT TGT GCT C-3′) or H624A, H628A mutation in NAP334 (rp 5′-CCG GGG ATC CTT AAT GAT TAT TTA GTT TGT TTG ATA AGT CCA TCG CTA TGG CGTA ATC GTT GGC CTC TAC AGG-3′) ubz mutations in CaRad30 were generated. But inverse PCR reaction was carried out by using primers NAP365 (5′-GCT CTT CAA GAG GCT GCA GAC TAT GCT TTA GCA TTG-3′) and NAP366 (5′-CAA TGC TAA AGC ATA GTC TGC AGC CTC TTG AAG AGC-3′) for generating ScRad30 with H568A, H572A mutations. CaRad30 F485A, L486A mutant was generated by replacing 1 to 1464 bp wild-type fragment with PCR product amplified using primers NAP03 and NAP231 (5′-GTG TTG AAT TCC GCG GCC TCT CTT AAT AAG ACA CAG-3′) by digesting with KpnI and EcoRI. All these amplified PCR products were purified, digested with BamHI, and cloned into a similar site in a 2μ-based vector with S. cerevisiae ADH1 promoter . Similarly, the ubz sequence from CaRad30 was fused to ScRad30 lacking the complete C-terminal domain including its own ubz and pip motif. PCR was performed with Q5 high-fidelity DNA polymerase using primers NAP286 (fp 5′- GGC CGG ATC CGT ATG TCA AAA TTT ACT TGG-3′) and NAP368 (rp 5′-GGC CGG ATC CTT AAT GAT TAT TTA GTT TGT TTG ATA AGT CCA TCG CTA TGT GGT AAT CGT TGT GCT CTA CAG GAT CGT CTA CAC TAA GTT TGC ATC TCG AAC ATT CCA ACT TCG G-3′) on Inverse PCR was carried out using the primer set NAP224 (5′-GTG AAT ATT CTT TAA AAT TAA TGG ATA TTG ATT CTG AAT TTG CAC AAG CTG ATG ATA TGG-3′)-NAP291 (5′-CCA TAT CAT CAG CTT GTG CAA ATT CAG AAT C-3′) on pUC19-CaPCNA to generate CaPCNA-79 . CaPCNA-90  was generated by swapping the mutated PCR product amplified using primers NAP31 (5′-GGC CAA GCT TGG ATC CAC ATA TGT TAG AAG GTA AAT TTG AAG-3′) and NAP225 (5′-G GCC GAA TTC GGA TCC CTA CTC ATC ATC ATC GAA TGC TGC TGC CAA G-3′) in pUC19-CaPCNA template. The PCNA mutated orfs were further subcloned to generate bacterial expression systems either with amino-terminal GST or carboxyl-terminal His-tagged CaPCNA mutants, similar to CaPCNA expression constructs .C. albicans strain is an auxotrophic isogenic derivative of SC5314. Deletion of the RAD30 gene in BWP17 was carried out as in the SC5314 strain of C. albicans (RAD30 deletion cassettes with a nourseothricin marker (pNA1389 and pNA1451) were generated, containing different lengths of upstream sequences but the same downstream gene sequence to facilitate an efficient homozygous knockout. Two successive rounds of transformation, followed by curing of the nourseothricin marker by maltose, resulted in the homozygous deletion of C. albicans strain CNA11. To express wild-type CaRad30, CaRad30 (1–601), and CaRad30 H624A, H628A mutants under repressible MET3 promoter, various constructs were integrated into the RP10 locus of CNA11 strain, and the transformants were selected by URA3 marker, and their expression was induced in the absence of methionine in synthetic dropout media.The BWP17 albicans . BrieflyC. albicans Polη was aligned with that of ScPolη by using pairwise sequence alignment tool, EMBOSS Needle (http://www.ebi.ac.uk/Tools/psa/emboss_needle/) to map the pip motif. Ubz model structures for CaRad30 and ScRad30 were generated by using FFAS (http://ffas.burnham.org/ffas-cgi/cgi/ffas.pl) online modeling database. The ubz domain of HsPolη with PDB ID 3WUP was used as a template for modeling. Secondary structure information was derived by observing the generated model.Seventy-seven Polη amino acid sequences from various organisms were taken from the NCBI database and aligned using the t-coffee multiple alignment tool. Further, they were segregated into three groups based on homology and presence or absence of critical pip/ubz motifs and again aligned to get final alignments. The amino acid sequence of E. coli BL21-DE3 strain as described earlier  and 150 mM NaCl at 4 °C to ensure complete removal of DTT and glycerol from the protein storage buffer, which could affect the heat exchange while they interact. ITC assays were performed using a Malvern MicroCal PEAQ-ITC calorimeter. Before the experiment, the cell and the syringe were thoroughly washed with water, followed by cell rinsing with a buffer. A control run was carried out to make sure that the buffer is not participating in heat change where the cell was filled with 300 μl of a buffer and concentrated CaPCNA or ScPCNA protein (200 μM) in the syringe. ITC was performed using various Polηs or UBZ peptide or UBZ mutant peptide in the sample cell and CaPCNA or ScPCNA or a mixture of CaPCNA+ WT UBZ peptide in the syringe. Twenty-five injections of 1.5 μl of protein from the syringe were given at intervals of 120 s with an initial delay of 120 s at 25 °C. The data were analyzed to determine the various kinetic parameters using a single-site binding model provided in the ITC analysis software package. The experiments were repeated twice for the positive interactions, whereas for the negative interactions, those were repeated multiple times at various concentrations of protein or peptide in the cell to confirm that there is no binding.GST-fused wild-type or mutant Polη proteins bound to glutathione sepharose beads were mixed with 0.5 μg of His-tagged wild-type or mutant CaPCNA, and a pull-down experiment was carried out using a standardized protocol, described previously . Then thFor size-exclusion chromatography, about 10 μg of purified proteins (CaPCNA and CaPolη) was incubated together at room temperature for 60 min, loaded onto a Superdex 200 PC3.2/30 column preequilibrated with a buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl and 10% glycerol. Chromatography was performed on an AKTA pure M system  at a flow rate of 0.05 ml/min at 4 °C, and the absorbance was monitored at 280 nm.rad30Δ and rad30Δ rev3Δ genomic knockout strains of S. cerevisiae harboring either wild-type or various mutant Rad30s were grown in dropout medium lacking Uracil (SD-Ura) to maintain selection for the plasmid. Knockout strains with empty vector YEplac195-ScADH1p  were also grown as a control. Cultures were grown up to the mid-logarithmic phase, washed with water, and resuspended to a density of 2 × 108 cells/ml. Desired dilutions of cells were spread onto the surface of SD-Ura plates for viability determination and onto SD-Arg plates containing canavanine for mutagenesis assays  length was performed for each strain using ImageJ software. All the graphs were made using the GraphPad Prism 8.0 software.Overnight grown culture of the different strains of C. albicans and resolved by alkali agarose gel electrophoresis as described earlier  overnight at room temperature prior to running. An equal volume of DNA was loaded for each sample into the wells and allowed to separate at 0.25 V/cm until the dye migrated to 60% of the length of the gel. Agarose gel was neutralized in 500 ml of 0.5 M Tris-HCl, pH 8.0 at room temperature for 1 h, and finally, the gel was stained with ethidium bromide, and the image was captured in a Chemidoc Imaging system (Bio-Rad).The total genomic DNA was isolated from different strains of  earlier . BrieflyC. albicans cells expressing different forms of Polη were grown in nonselective liquid medium (YPDU) to an OD600 of ∼1, and the serial dilutions of their cells  were spread onto SD+5′- FOA (1 μg/μl) and YPDU plates. After plating, one of the sets of plates was exposed to 16 J/m2 UV and wrapped in aluminum foil. All the plates were incubated for 3 to 4 days at 30 °C. FOA-resistant colonies were counted, and some of the resistant colonies were picked up and restreaked on SD-Ura, SD-Arg, and SD-His plates to ascertain the genotypes.All data presented is contained within this manuscript.This article contains The authors have no conflicts of interest to declare regarding the publication of this article.


WW domain-containing oxidoreductase (WWOX) is known as one of the risk factors for Alzheimer’s disease (AD), a neurodegenerative disease. WWOX binds Tau via its C-terminal SDR domain and interacts with Tau phosphorylating enzymes ERK, JNK, and GSK-3β, and thereby limits AD progression. Loss of WWOX in newborns leads to severe neural diseases and early death. Gradual loss of WWOX protein in the hippocampus and cortex starting from middle age may slowly induce aggregation of a protein cascade that ultimately causes accumulation of extracellular amyloid beta plaques and intracellular tau tangles, along with reduction in inhibitory GABAergic interneurons, in AD patients over 70 years old. Age-related increases in pS14-WWOX accumulation in the brain promotes neuronal degeneration. Suppression of Ser14 phosphorylation by a small peptide Zfra leads to enhanced protein degradation, reduction in NF-κB-mediated inflammation, and restoration of memory loss in triple transgenic mice for AD. Intriguingly, tumor suppressors p53 and WWOX may counteract each other in vivo, which leads to upregulation of AD-related protein aggregation in the brain and lung. WWOX has numerous binding proteins. We reported that the stronger the binding between WWOX and its partners, the better the suppression of cancer growth and reduction in inflammation. In this regard, the stronger complex formation between WWOX and partners may provide a better blockade of AD progression. In this review, we describe whether and how WWOX and partner proteins control inflammatory response and protein aggregation and thereby limit AD progression. Alzheimer’s disease (AD) is the most common cause 60–75%) of dementia and an age-related neurodegenerative disease. Occurrence of AD is frequently seen in patients over 65 years old—the so called late-onset AD. For early-onset AD, the symptoms may start in patients in their 30s or 40s due to gene mutations inherited in an autosomal dominant fashion ,2,3. The% of demeExtracellular amyloid beta (Aβ) plaques around the neurons and intracellular neurofibrillary tangles (NFTs) caused by hyperphosphorylated tau proteins are considered the key markers of AD ,2,3. SigPathogenic tau, Aβ, and other proteins can be spread by transmitting from one neuron to another by exocytosis or via synapses or extracellular vesicles (EV) in the central nervous system (CNS), or transferring to the CNS from the peripheral nervous system ,11,12,13It appears that hyperphosphorylated tau protein captured in the EV is conformationally altered and is pathogenic. Tau binds and stabilizes microtubules. A small portion of intracellular tau is released to the extracellular space under physiological conditions . Tau is Presumably, tau undergoes misfolding during the period from middle to old age, and then the misfolded proteins are transmitted among neurons prior to the appearance of AD symptoms. Conceivably, misfolded tau proteins are the pathological seeds for native tau to merge and become misfolded. Phosphorylation in many specific amino acid residues in tau causes tau aggregation. Acetylated tau promotes memory loss due to disruption of the formation of spatial memories . TargetiC. elegans, no significant aggregation of amyloid β occurs [There is an age-dependent increase in protein instability and aggregation ,17,18. Tβ occurs ,5,6,7.The upstream pathway(s) that causes Aβ aggregation is largely unknown. Furthermore, the deciding factors rendering protein aggregation are not well defined. If protein aggregation starts in middle age and goes on very slowly year by year, it would be very difficult to identify and characterize the aggregation initiator(s) and the downstream protein aggregation cascade(s). Aggregated protein complexes suppress proteasomal function , and indTransforming growth factor beta (TGF-β) orchestrates the formation of extracellular matrix and controls cell growth or death. TGF-β strongly inhibits the growth of epithelial and endothelial cells ,21,22. TWe have demonstrated the presence of TGF-β-regulated protein aggregation in the hippocampi of middle-aged normal human brains. Three proteins have been identified, which are TGF-β-induced antiapoptotic factor (TIAF1), trafficking protein particle complex subunit 6A (TRAPPC6A or TPC6A), and SH3 domain-containing GRB2 such as endophilin B2 (SH3GLB2) ,28,29,30Transiently overexpressed TIAF1 polymerizes and causes degradation of membrane amyloid precursor protein (APP), followed by generation of Aβ and amyloid fibrils in vitro ,28,29,30In the following sections, we will discuss how tumor suppressor WW domain-containing oxidoreductase  limits protein aggregation ,35,36,37N-terminal WW domains containing conserved tryptophan residues, a nuclear localization signal located between the WW domains, a C-terminal short-chain alcohol dehydrogenase/reductase domain (SDR), and a proapoptotic C-terminal tail termed D3 [N-terminal first WW domain binds PPxY or PPPY motif in the target proteins [WWOX protein possesses two ermed D3 ,38,39,40ermed D3 ,35,36,37proteins ,33,39,40Second, when cells are exposed to stress stimuli, WWOX undergoes Tyr33 phosphorylation  Figure . pY33-WWN-terminal WW domain and the C-terminal SDR domain [Zfra (Zinc finger-like protein that regulates apoptosis) is a 31-amino-acid protein that binds WWOX at both the R domain . Zfra isR domain . pY33-WWR domain . The binR domain . In contR domain . The difThird, the SDR domain contains a catalytic site to bind NADPH and contributes to the redox activity of WWOX, which is involved in aerobic metabolism ,34,35,36Finally, tyrosine kinase ACK1 induces polyubiquitination of WWOX to undergo proteasomal degradation , which iA portion of WWOX is located in the cell membrane ,51,56,57Wwox deficiency leads to neurodevelopmental and degenerative neuropathies in newborns [Wwox knockout and heterozygous mice are readily subjected to seizure attack [Neuronal heterotopia or neuronal migration disorder contributes, in part, to the development of seizures ,59,60, anewborns ,62,63,64newborns . Both Wwe attack . These me attack .WWOX/Hyal-2 signaling is involved in traumatic brain injury (TBI). During TBI in rats, Hyal-2 and WWOX undergo nuclear translocation, and the Hyal-2/WWOX complex becomes accumulated in the nucleus and causes neuronal death . In vitr2+ influx in response to UV or apoptotic stress at room temperature [2+ influx. WWOXd cells undergo non-apoptotic explosion in response to UV irradiation in room temperature.In principle, WWOXf cells exhibit BCD and functional Caperature . When ceperature . Hyaluroperature ,50. HoweIn vitro studies showed that pro-survival JNK1 blocks the proapoptotic function of WWOX ,68. DuriTransiently overexpressed WWOX frequently sequesters transcription factors in the cytoplasm, and thereby blocks their transcription for prosurvival proteins in the nucleus of cancer cells in vitro ,34. HoweC-terminal SDR domain [WWOX blocks Tau hyperphosphorylation by directly binding Tau via its R domain ,69. By dR domain ,69. In cR domain ,69. TogeR domain .WWOX/Wwox gene causes severe neural diseases , metabolic disorders , and early death in newborns [WWOX-related epileptic encephalopathy (WOREE syndrome) [During mouse embryonic development, WWOX protein is highly expressed in the neural crest-derived structures such as cranial and spinal ganglia, skin pigment cells, and mesenchyme in the head, indicating the potential involvement of WWOX in neuronal differentiation and maturation ,75,76,77newborns ,73,74,75newborns . Togetheyndrome) ,71,72,73yndrome) . ExtensiWe reported that when WWOX protein is downregulated in the hippocampi of middle-aged individuals, brain protein aggregation may start to occur ,5,6,7. TWwox knockout mouse, it only takes less than 15 days after birth to let the brain proteins polymerize and aggregate in a cascade-like manner [Wwox heterozygous mice, they exhibit a faster kinetics of AD progression, compared to triple-transgenic mice [In e manner ,26,28,75Under physiological conditions, TPC6A or TPC6AΔ binds the C-terminal tail of WWOX in the cytoplasm and provides a piggyback ride for WWOX to translocate to the nucleus . The WWOWWOX prevents the aggregation of TPC6A or TPC6AΔ in the cytoplasm. Without the presence of WWOX, the fluid-phase TPC6AΔ acquires S35-phosphorylation, binds mitochondria, and undergoes polymerization. TPC6AΔ binds membrane-bound pS37-TIAF1 on the mitochondrial surface. The TPC6AΔ/TIAF1 complex stimulates activation of caspase 3, which leads to Thr688-dephosphorylation of the membrane amyloid precursor protein (APP). APP is then degraded to generate an intracellular domain (AICD) and Aβ. Aβ is secreted into the extracellular matrix to form amyloid fibrils and plaques ,6,75.When mice are subjected to traumatic brain injury or sciatic nerve dissection, endogenous WWOX is significantly upregulated and phosphorylated at Tyr33 ,42,50,80When pY33-WWOX is downregulated, pS14-WWOX is significantly increased in the lesions of AD hippocampus and cortex ,28. pS14The aforementioned pY33-pS14 transition raises the question regarding whether binding of WWOX with its protein partners contributes to the control of disease progression. We determined that Zfra4-10 or WWOX7-21 peptide strongly suppresses the growth of cancer xenografts in mice ,58. The In contrast, when Zfra4-10 and WWOX7-21 peptides are in combination in treating mice, binding of endogenous WWOX with protein partners is significantly reduced in many organs, and the cancer xenograft growth is increased .N-terminal WW domain and C-terminal SDR domain of WWOX [Finally, Zfra4-10 covalently interacts with proteins for rapid degradation in a ubiquitination/proteasome-independent manner ,81. Zfra of WWOX . Conceiv of WWOX .Tumor suppressor p53 participates in the AD progression and neurodegeneration ,83,84. Hhttps://hbatlas.org/pages/hbtd; last visit on 25 June 2021) [We selected 16 WWOX-related gene expression data from the Human Brain Transcriptome (ne 2021) ,86. Braine 2021) , no redune 2021) . No downWWOX signals with p53, Smad4, and HYAL-2 for growth suppression, apoptosis, and BCD ,36,55,76Both p53 and WWOX are guardians of the genome ,33,87,88When cells are exposed to micromolar amounts of 17-beta estradiol (E2), cell death occurs in a p53/WWOX-dependent manner . MechaniN-terminal WW domain of WWOX. Activated p53 binds TIAF1, and WWOX strengthens the p53/TIAF1 complex so as to stabilize the triad. Without p53 activation, no binding interaction occurs between TIAF1 and p53 [TIAF1 complexes with p53 and WWOX to form a triad . The TIA and p53 . Silenci and p53 . When TI and p53 , suggestWhen extracellular matrix is altered in the microenvironment, intracellular TIAF1 is upregulated and undergoes aggregation. For example, when any cells are seeded on extracellular matrices derived from other cells, these cells start to increase the intracellular levels of aggregated TIAF1 and Aβ ,27,91. TPolymerized TIAF1 binds Smad4 and blocks SMAD promoter activation . When p5While p53 and WWOX act synergistically in vitro in inducing apoptosis, it is intriguing to find that p53 may functionally antagonize with WWOX in vivo . This fuWhen in combination, p53 strongly abolishes the biological effects of WWOX, including cancer suppression and inhibition of inflammation . Most strikingly, cancer-regulated protein aggregation in the brain is blocked by WWOX . Again, Despite more than 100 years of research in AD, there are still no effective therapeutic drugs available to cure the disease ,3,7. NowIt is generally considered that there is an inverse relationship between the development of cancer and neurodegeneration in vivo ,93. It iOne of the proteins that runs in parallel in promoting cancer and neurodegeneration is pS14-WWOX ,81. pS14WWOX exhibits a plethora of functional properties in vivo. WWOX restricts cancer growth and AD progression, induces immune cell differentiation ,53,54, aUnfortunately, pS14-WWOX is significantly upregulated in the lesions of cancer  and AD hWwox heterozygous mice [Zfra4-10 and Zfra1-31 peptides are of therapeutic potential in blocking AD progression . Two potous mice . The fulous mice ,95. As sous mice ,81. SimiSecond, exogenous full-length Zfra1-31 and Zfra4-10 peptides induce memory anticancer response via expansion of Zfra-reactive spleen Z lymphocytes ,57,81. WSupporting evidence showed that the stronger the binding strength between WWOX and its partner proteins, the better the extent of cancer suppression . By the In conclusion, WWOX and its binding proteins play a critical role in limiting inflammation and associated AD progression. The stronger the binding, the better the inhibition of inflammation and suppression of AD symptoms. Presumably, when there is an elevated intracellular Zfra, Zfra binds WWOX and the Zfra/WWOX complex is subjected to degradation. Consequently, free, excessive p53 and other WWOX-binding proteins start to exert inflammatory reactions ,76.Despite WWOX being a defined risk factor for AD , functioEctopic p53 and WWOX mediate apoptosis in a synergistic manner in vitro ,53,80, wDespite several decades of global efforts, there are still no effective drugs available in curing AD. A recent monoclonal antibody-based drug Aducanumab for AD treatment has been approved by FDA . Nonethe


The folding of many proteins can begin during biosynthesis on the ribosome and can be modulated by the ribosome itself. Such perturbations are generally believed to be mediated through interactions between the nascent chain and the ribosome surface, but despite recent progress in characterising interactions of unfolded states with the ribosome, and their impact on the initiation of co-translational folding, a complete quantitative analysis of interactions across both folded and unfolded states of a nascent chain has yet to be realised. Here we apply solution-state NMR spectroscopy to measure transverse proton relaxation rates for methyl groups in folded ribosome–nascent chain complexes of the FLN5 filamin domain. We observe substantial increases in relaxation rates for the nascent chain relative to the isolated domain, which can be related to changes in effective rotational correlation times using measurements of relaxation and cross-correlated relaxation in the isolated domain. Using this approach, we can identify interactions between the nascent chain and the ribosome surface, driven predominantly by electrostatics, and by measuring the change in these interactions as the subsequent FLN6 domain emerges, we may deduce their impact on the free energy landscapes associated with the co-translational folding process. NMR measurements of methyl relaxation in translationally-arrested ribosome–nascent chain complexes probe the dynamics of folded nascent polypeptides emerging during biosynthesis and quantify their interaction with the ribosome surface. Through8 bL12 was expressed and purified as previously described.11 FLN5 RNC samples were expressed and purified following an adaptation of previous protocols:8,9E. coli BL21 (DE3) cells were adapted progressively into MDG media  and then grown to an OD600 of 3 . Cells were resuspended in an equal volume of EM9 media -butyric-4-13C acid). Following 30 min incubation , expression was induced with 1 mM IPTG for 50 min at 37 °C. Cells were then harvested and RNCs purified using previously reported protocols.7 Purified RNCs were resuspended in Tico buffer  EDTA-free protease inhibitor tablet (Roche), pH 7.5), flash frozen and stored at −80 °C.Wild-type FLN5 and variants were expressed and purified as previously described.2H, 13CH3-ILV]-labelled 70S ribosomes were prepared from E. coli BL21 (DE3) cells grown in MDG media -butyric-4-13C acid) at 37 °C and harvested at an OD600 of 3. Ribosomes were purified following the same protocol as RNCs, omitting the metal affinity chromatography step.Unprogrammed  labelled FLN5 was prepared at 500 μM in Tico buffer , and sidechain resonances were assigned based on a previous backbone assignment8 using H(CCO)NH, (H)C(CO)NH, H(C)CH and (H)CCH TOCSY experiments. U- labelled bL12 was prepared at 300 μM in Tico buffer, and sidechain resonances were assigned based on a previous backbone assignment19 using a 4D HC(CCO)NH TOCSY experiment.U--labelled FLN5 was prepared at ca. 100 μM in deuterated Tico buffer  containing 0, 40, 50 and 60% (w/w) d8-glycerol. 1H R2 relaxation measurements were acquired for each sample using an adapted methyl-SOFAST HMQC experiment , bL12 resonances were instead used to monitor sample integrity. K5 RNC samples were treated with 60 μg ml−1 RNase A to release the NC following acquisition.1H R2 rates in the K5 RNC were estimated by comparison of the spectrum noise level (as an upper limit on peak intensities), σK5, relative to peak intensities in the wild-type FLN5+67 spectrum, IWT . 1H and 13C linewidths for the wild-type NC  were determined using NMRPipe. Accounting for the effects of line widths and relaxation during the pulse sequence on the intensity of cross-peaks, the relative intensity in the two experiments is:22R2 = RK52−Rwt2 and τ = 1/2JCH = 4 ms. From this expression, lower bounds for RK52 = Rwt2 + ΔR2 could be determined. To reduce the sensitivity to changes in the 13C relaxation rate, which we assume here to be comparable to the change in the 1H relaxation rate, both spectra were processed with a strong 150 Hz exponential window function in the indirect dimension .Lower bounds for 13CH3 labelling of isoleucine Cδ methyl groups.8 However, as this provides rather sparse coverage across the domain, to enable a more detailed structural analysis here we have expanded our labelling approach to include leucine and valine methyl groups, which are widely distributed throughout the domain  than in the isolated protein  . As the effective rotational correlation time, τc, of the bL12 CTD has previously been determined as ca. 14 ns,22 this provides an approximate lower bound for the mobility of the NC.To investigate the mobility of the NC we then acquired s Fig. S3. These my Fig. S4. The meay Fig. S4 and S5†, ± s.d.) , indicatSaxis2τc, where Saxis2 is an order parameter for the three-fold methyl symmetry axis, representing motion on a ps–ns timescale, and τc is an effective rotational correlation time, which may depend on the orientation of the methyl symmetry axis relative to the rotational diffusion tensor, as well as transient interactions with the ribosome surface  between Saxis2τc values in isolated FLN5 and in the NC , and no significant differences were found with respect to the WT FLN5+67 RNC. This is presented in terms of estimated Saxis2τc values in τGSc/τWTc = 1.07 ± 0.03 (r = 0.85 ± 0.13). Thus, we conclude that neither the residual structure, compaction or interactions of the linker region are responsible for the reduced mobility of the FLN5 domain within RNCs.We first explored the effect of substituting the FLN6 linker for a poly-GS sequence that is neutral, non-hydrophobic, disordered, and that has previously been shown not to alter the onset of co-translational folding,Z = −9) on electrostatic interactions with the predominantly negatively charged ribosome surface.31 We have previously introduced the E6 variant for the analysis of interactions of the unfolded state,7 in which six surface residues were substituted with negatively charged glutamate residues  and K5 , in which a second cluster of surface residues were mutated to positively charged lysine residues , from which the relative changes in mobility may be determined. As discussed earlier for the WT RNC, such linear correlations are consistent with transferred relaxation from a bound state, and the reduction in mobility as the net charge on the NC decreases indicates the role of electrostatic interactions between positively charged patches on the NC and the negatively charged ribosome surface. However, given that the WT and E6 variant also exhibit similar behaviour   . An incrtunnel7) , consist2O at 298 K) is 7.7 ns,29 and the ribosome itself is ca. 3.6 μs,30 complete binding would correspond to a change in effective correlation time τriboc/τisoc ≈ 430. Thus, for the wild-type RNC from linker lengths of 47 to 110  rotational correlation time of the isolated domain (in D7 to 110 , we esti7 This is plotted here in terms of the free energy of interaction,  (Gbound-free on linker length is similar for observed lengths of native and unfolded states, indicating that the changing linker modulates the effective concentration of the ribosome surface to a similar extent. However, interactions of the unfolded state are approximately 2 kcal mol−1 stronger than for the native state, corresponding to a ca. 30-fold difference in association constant. We may combine these data with previous measurements of co-translational folding energetics,10 to construct a free energy diagram illustrating for the first time the impact of interactions of both folded and unfolded states on the co-translational folding of a NC (The interactions we observe here can be compared with the much stronger interaction of a C-terminal segment within unfolded NCs, which has a bound population of up to 90% at short tether lengths.action,  . The dep of a NC .7−1,7 without perturbing the interactions of the native state; but equally, interactions of the native state can be increased to at least ca. 25% within the K5 variant (We have previously shown that the effect of an interaction on the net energetics of folding is:nd state , do not  variant . Therefo4,5,8,32–34 This is usually ascribed to the effect of interactions, although given the diversity of the proteome, such interactions are likely to be variable in strength and significance. Ultimately, the ability to predict the strength of these interactions would be valuable in better understanding the general mechanisms of co-translational folding. The measurements we have undertaken in this work, together with analogous measurements of the unfolded state,7 provide initial steps in this direction. By determining the first complete description of interactions for both sides of the folding equilibrium, we may begin to progress studies of co-translational folding towards a quantitative standard comparable to that achieved for the reversible folding of isolated domains.The ribosome has now been shown to perturb the co-translational folding of many different proteins, generally leading to a reduction in stability relative to the isolated domain.Relaxation and cross-correlation relaxation datasets supporting this article have been uploaded as part of the ESI.Conceptualization: CAW, JC. Funding acquisition: JC. Investigation: CB, CAW. Methodology: CAW, TW. Supervision: CAW, AMEC, LDC, JC. Writing – original draft: CB, CAW, JC. Writing – review & editing: all authors.There are no conflicts to declare.SC-012-D1SC04313G-s001


Neurodegenerative diseases are pathologies of the central and peripheral nervous systems characterized by loss of brain functions and problems in movement which occur due to the slow and progressive degeneration of cellular elements. Several neurodegenerative diseases are known such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis and many studies on the molecular mechanisms underlying these pathologies have been conducted. Altered functions of some key proteins and the presence of intraneuronal aggregates have been identified as responsible for the development of the diseases. Interestingly, the formation of the SNARE complex has been discovered to be fundamental for vesicle fusion, vesicle recycling and neurotransmitter release. Indeed, inhibition of the formation of the SNARE complex, defects in the SNARE-dependent exocytosis and altered regulation of SNARE-mediated vesicle fusion have been associated with neurodegeneration. In this review, the biological aspects of neurodegenerative diseases and the role of SNARE proteins in relation to the onset of these pathologies are described. Neurodegenerative diseases are a group of pathologies affecting the central and peripheral nervous system, they are characterized by a variety of clinical and pathological expressions and occur due to the loss of neurons in different regions ,2. NeuroThe classification of neurodegenerative disorders is challenging due to the high number of pathologies (a few hundred) and difficulty in making clear clinical and pathological distinctions . Severala, Qb, Qc and Qb-c SNAREs, depending on their location in the four helix-bundle and on how many SNARE domains they contribute with to the formation of the complex . Mo. Mo93]. One of the roles of SNARE proteins in PD is based on their interaction with one of the key molecules, α-syn, which is involved in the pathogenesis of PD. In fact, it has been demonstrated in vitro that large α-syn oligomers can bind to the N-terminal of Vamp2 ,65, and Interestingly, α-syn aggregation has been correlated with alterations of the secretory pathway and accumulation of toxic dopamine that occurred in the cytosol. Therefore, impairment of the trafficking between ER and the Golgi apparatus, subsequent Golgi fragmentation and cytoskeleton alterations were related to syntaxin-5 expression . InteresLRRK2), a large kinase protein with GTPase activity, with a role in synaptic vesicle fusion and cytoskeletal-related processes [Another gene associated with PD is leucine-rich repeat kinase 2  transport. Impairments of MVs trafficking have been associated with PD pathogenesis as mutations in ms of PD ,99,100. ms of PD ,102,103.ms of PD ,89. In pms of PD . Howeverms of PD .ALS is an adult-onset neurodegenerative disease characterized by motor system degeneration, progressive muscle atrophy and spasticity followed by respiratory failure and death . Moreove+ astrocytes was due to ER calcium overload and was linked to elevated ATP release which was inhibited by the over-expression of dominant-negative VAMP2. The release of toxic factors was correlated with disease onset [An important characteristic of neurodegenerative diseases is the presence of protein aggregates containing ubiquitin. Liu and colleagues have evaluated the role of ubiquitination in the functionality of the SNARE complex composed of VAMP2, SNAP-25 and syntaxin-1 in the brain and spinal cord of transgenic mice and how the alteration of this complex due to the modification of VAMP2 with a “noncleavable” N-terminal ubiquitin substrate could lead to adult-onset paralysis and neurodegeneration . This SNse onset .2+-mediated fusion of vesicles with the presynaptic membrane, are normally responsible for the onset and progression of the disease and SNARE proteins are fundamental components of the machinery that allows membrane fusion and neurotransmitter release at the axon terminal [Degeneration of neurons and other cellular elements to them associated leads to neurodegenerative diseases such as AD, PD, ALS, HD and SMA. One important aspect of these pathologies is the dysregulation of the vesicle trafficking by alterations of the proteins responsible for exocytosis, endocytosis and neuronal survival. On one hand, the death of neuronal cells begins with the distal part of the axon and then the cell body is affected. Impairments of key steps of vesicle exocytosis, such as docking, priming, and Caterminal ,68. On tterminal ,44,56. T