The C-terminal domain half protein with the quadruple mutation, like the wild-type one, could interact with the N-terminal domain or the CP47 E-loop in vitro. The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38, but distinct from the wild type. The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38. We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane, thus impairing its function in chloroplast.Arabidopsis cyclophilin38 (CYP38) is a thylakoid lumen protein critial for PSII assembly and maintenance, and its C-terminal region serves as the target binding domain. We hypothesized that four conserved residues  in the C-terminal domain are critical for the structure and function of CYP38. In yeast two-hybrid and protein pull-down assays, CYP38s with single-sited mutations  did not interact with the CP47 E-loop as the wild-type CYP38. In contrast, CYP38 with the R290A/F294A/Q372A/F374A quadruple mutation could bind the CP47 E-loop. Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of  FK506 binding proteins (FKBPs) and cyclophilins (CYPs) are cellular receptors for immunosuppressant drugs FK506 and cyclosporine A, respectively . They arIn Arabidopsis, a large number of immunophilins are present in chloroplast: 1 CYP in the stroma, 5 CYPs and 11 FKBPs in the thylakoid lumen . The strThe PPIase activity of immunophilins was initially thought to be essential for their physiological roles, however, it was found that there is no tight correlation between the PPIase activity and their functions . In Arabcyp38 mutant  are important for the structure and function. To exam this possibility, we used a site directed mutagenesis approach to create alternations in these conserved residues. Then we tested impact of these changes on the structure of CYP38 All the primers used in this study are listed in cyp38 mutant (Garlic 206 38), and transgenic plants used in this study are all in the background of the Columbia ecotype. Generally, plants were grown on soil at 25°C under low light (30 μmol m–2 s–1) for 14 days, then subjected to continuous illumination (80–100 μmol m–2 s–1), similar to growth conditions described earlier (CYP38 were created as following. The full length coding sequences (CDSs) of wild type and mutated CYP38s were cloned behind the cauliflower mosaic virus (CaMV) 35S promoter in the binary vector pRI101-AN, and the constructs were transferred into Agrobacterium tumefaciens strain GV3101 and introduced into cyp38 via by the floral dip method . The primers are listed in DpnI  for 2 h at 37°C, and 5 μL of the reaction was used to transform E. coli DH5a competent cells. The resulting vectors were confirmed by DNA sequence analysis.Basically, the procedure as described before was followed . The fulTotal leaf proteins were extracted by grinding leaf material and resuspended in 2x SDS loading buffer  SDS, 2% (v/v) 2-mercaptoethanol, 0.001% (w/v) bromphenol blue, 20% (v/v) glycerol), followed by incubation at 98°C for 10 min. Immunoblotting was performed as before . ProteinThe DNA sequences encoding wild type and mutated CYP38s were cloned into pGBKT7 and used as baits. The coding sequences of CP47 E-loop was cloned into pGADT7 and used as prey. Yeast transformants were selected on synthetic dropout medium lacking Leu and Trp (−LT). To screen protein–protein interactions, transformed yeast cells of different dilutions  were spotted onto the selection media lacking Leu, Trp, Ade, and His (−LTAH), and the cell growth was used as an indicator of protein-protein interaction .CP47 E-loop cDNA was cloned downstream of the His tag in the expression vector pET-28a (+). All constructs were introduced into the E. coli BL21 (DE3) strain, and recombinant protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactoside at 20°C for overnight. The GST fusion proteins and His fusion protein were purified through affinity chromatograph with Glutathione sepharoseTM 4B  and Ni-NTA Agarose  according to the manufacturer’s instruction, respectively. The in vitro pull-down assay was performed as previously described with minor modifications  for 2 h at 4°C. The beads were washed four times with washing buffer , and the proteins were eluted with 2x SDS buffer, boiled for 10 min, and subjected to Western blotting with anti-GST antibody and anti-His antibody, respectively.The DNA sequences encoding wild type and mutated CYP38s were cloned downstream of the GST tag in the expression vector pGEX-4T-3, and ications , Ni-NTA 3, and 0.1% BSA). Homogenates were then filtered through 2-layer gauze. Stromal, thylakoid lumen, and membrane fractions were prepared as previously described with slight modifications (2) were overlaid on Percoll step gradients [40 and 80% (v/v)] and were centrifuged at 4,000 g for 20 min. Collected chloroplasts at a chlorophyll concentration of 0.5 mg mL–1 were solubilized in lysis buffer  for 10 min on ice. The stromal and membrane fractions were separated by centrifugation at 18,800 g for 10 min at 4°C. The supernatant containing the stroma subfraction was collected and pellets were solubilized with 0.2% digitonin in the lysis buffer for 20 min on ice. After centrifugation at 18,800 g for 1 h at 4°C, thylakoid lumen proteins were collected from the supernatant and the membrane proteins in the pellet were resuspended in resuspension buffer.Leaves from 4-week-old seedlings were ground in a blender with ice-cold homogenization buffer . Homogenates were then filtered through 2-layer gauze. After centrifugation at 2,000 g for 8 min at 4°C, the pellet was resuspended in SH buffer . Thylakoid membranes were isolated by centrifugation at 2,400 g for 2 min at 4°C. Pelleted membranes were resuspended in SH buffer and was used for additional analyses.Thylakoid membranes were isolated from 4-week-old seedlings as described previously with minor modifications . Leaves –1 chlorophyll. Thylakoid membranes were solubilized with 1% (w/v) n-dodecyl-β-D-maltoside or 4% (w/v) digitonin (Sigma-Aldrich) for 10 min on ice. After centrifugation at 10,000g for 10 min, the supernatant was mixed with 1/10 volume of BN sample buffer  sucrose). Then, samples were loaded onto blue native 5–13.5% polyacrylamide gradient gel. Electrophoresis was performed at 100 V in 4°C. The cathode buffer initially contained 0.01% Serva Blue G dye  and was replaced by buffer lacking dye after approximately half of the electrophoresis run. After electrophoresis, each lane was excised from the gel and incubated in 2x SDS buffer. Then, proteins were separated using SDS-PAGE on a 12% polyacrylamide gel. After electrophoresis, the gels were used for immunoblot analysis  to a final concentration of 1 mg mLanalysis .The residues R290, F294, Q372, and F374 in the C-terminal domain (239-437) of Arabidopsis CYP38 are highly conserved . Becausein vitro.The C-terminal domain of CYP38, but not the mature full-length CYP38, could interact with its target, the CP47 E-loop because the N-terminal domain associates tightly with the C-terminal domain and blocks its access to the CP47 E-loop . If a chAs expected, YTH assays showed that the C-terminal domain of CYP38 (C-domain) interacted well with the CP47 E-loop, while the full length CYP38 did not . When thin vitro YTH and protein pull-down experiments, we further tested functional effects of those mutations in planta. Full-length CYP38 cDNAs with designed mutations driven by the 35S promoter were introduced into the Arabidopsis cyp38 mutant, which is the cyp38-2 null allele used in a previous study  had a stunted growth, but less pronounced than cyp38 . To explore this possibility, we included the C-domain protein (239–437) and its mutant form with the quadruple mutation (R290A/F294A/Q372A/F374A-C) in the yeast two-hybrid assay, and tested their interaction with the N-domain protein (92–215). The result showed that the mutated C-domain, as well as the wild-type C-domain, could interact with the N-domain , suggestBecause CYP38 with the quadruple mutation could interact with the CP47 E-loop, this raised the possibility that the mutation might influence the interaction between the C-domain and the CP47 E-loop. However, both yeast two-hybrid and protein pull-down assays showed that the mutated C-domain interacted with the CP47 E-loop as the normal C-domain . It thusin vivo, and therefore it might impair the association of CYP38 with the thylakoid membrane. This possibility was tested by fractionating isolated chloroplasts into stroma, thylakoid lumen and thylakoid membrane, and determining the sub-chloroplast localization of CYP38 by immunoblotting. The ClpC, PC, and D1 proteins were used as markers for stroma, thylakoid lumen, and thylakoid membrane, respectively. Their profiles demonstrated that the chloroplast fractionation was successful  analysis was used to determine how the mutation might influence PSII assembly. Purified thylakoid membranes of wild type, cyp38, and quadruple mutation plants were treated with N-dodecyl-β-D-maltoside (β-DM) and resolved by BN-PAGE analysis. The cyp38 plants were almost completely devoid of PSII SCs which were obviously present in wild-type samples  could be important for the protein structure and function, even if they are not related to PPIase activity. Each single mutation at these four sites did not affect the of CYP38 , showingin vitro , suggestt plants .R290 and F294 are located at the β5 sheet, while Q372 and F374 are just in front of the β7 sheet, and both sheets could be very important for the structural integrity of the C-terminal domain . We compin vitro assay.Because CYP38 with the quadruple mutation interacts with the CP47 E-loop, we assume that the intramolecular N-C interaction in the mutated CYP38 is weaker than that in wild type. Therefore the CP47 E-loop would prevail in the competition with the N-domain of CYP38, and bind the full length mature CYP38 protein. However, we found that the mutant C-domain half protein still interacted with the N-terminal half protein in YTH assays, similar as its wild-type counterpart . It is pin planta and in vitro experiments could be explained that the in vitro experiments were not sensitive enough to reflect processes occurring in planta.The membrane association assay showed that CYP38 with the quadruple mutation associated less with thylakoid membrane complexes than the wild-type CYP38 . BN-PAGEin vivo.In conclusion, we consider that the conserved amino acids examined in this study are critical for CYP38 to maintain its dynamic intramolecular N-C interaction and the ability to bind its target proteins. Mutations in those sites could weaken the N-C interaction and diminish its regulatory mechanism in response to changing light conditions. The mutated CYP38 could still recognize and bind its target proteins, but its alteration would affect its function fkbp20-2 mutant plants showed a stunted growth phenotype as well (We noticed that most of the thylakoid lumen immunophilins, except CYP20-2 and FKBP13, are divergent and have lost most of the conserved residues required for PPIase activity, similar to CYP38 . The rem as well . It is wThe original contributions presented in the study are included in the article/AF, YW, MX, and LS designed the research. LS, LD, JWe, XZ, WZ, and JWa performed the research. LS, YW, MX, and AF analyzed the data. LS, YW, and AF wrote the manuscript with contribution and approval from all authors.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA-binding proteins (RBPs) are a class of proteins known for their diverse roles in RNA biogenesis, from regulating transcriptional processes in the nucleus to facilitating translation in the cytoplasm. With higher demand for RNA metabolism in the nervous system, RBP misregulation has been linked to a wide range of neurological and neurodegenerative diseases. One of the emerging RBPs implicated in neuronal function and neurodegeneration is splicing factor proline- and glutamine-rich (SFPQ). SFPQ is a ubiquitous and abundant RBP that plays multiple regulatory roles in the nucleus such as paraspeckle formation, DNA damage repair, and various transcriptional regulation processes. An increasing number of studies have demonstrated the nuclear and also cytoplasmic roles of SFPQ in neurons, particularly in post-transcriptional regulation and RNA granule formation. Not surprisingly, the misregulation of SFPQ has been linked to pathological features shown by other neurodegenerative disease-associated RBPs such as aberrant RNA splicing, cytoplasmic mislocalization, and aggregation. In this review, we discuss recent findings on the roles of SFPQ with a particular focus on those in neuronal development and homeostasis as well as its implications in neurodegenerative diseases. Near identical DNA is present in every cell of an individual organism, yet it is the presence of protein factors and regulatory RNAs in transcriptional and post-transcriptional processing that are vital for producing the temporal and spatial cellular diversity observed . There aDrosophila behavior/human splicing (DBHS) protein family, a highly conserved group of nuclear proteins exclusively found in the animal kingdom . T. TMapt g protein  [53].sfpq mutant embryos while no loss of motoneurons was observed in the brain or in the spinal cord, indicating that SFPQ has specific roles in the differentiation of certain neuronal cell types [From a broader perspective, an earlier study of neurogenesis in zebrafish found that SFPQ is important for the survival and differentiation of neuronal cells . Zebrafill types .sfpq was discovered which was rescued by microinjecting human sfpq mRNA [Although SFPQ is found primarily in the nucleus, a small subset of the total cellular pool of SFPQ has been observed in the cytoplasm of motor axons and shown to drive axon maturation and connectivity . Using afpq mRNA . The autIn another study, colocalization of SFPQ/NONO with another abundant RBP, HERMES , and the stress granule component G3BP1 were observed when neuronal differentiation was induced in retinal ganglion cells, resulting in the formation of cytoplasmic neuronal granules . Colocallaminb2 and bclw in dorsal root ganglion sensory neurons to support axon survival [laminb2 and bclw mRNA from the nucleus to the axon by RNA transport granules. LaminB2 and Bclw proteins are then translated for the use of maintaining axonal survival and functions. When SFPQ was knocked down, the protein expression level of LaminB2 and Bclw in the axon decreased, leading to axon degeneration. This indicates the function of SFPQ in regulating the level of laminb2 and bclw mRNA in the axon for translation and in turn, supporting axonal survival [Similarly, SFPQ was also found to interact and guide multiple axonal mRNAs such as survival . It has survival .More recently, it has been demonstrated that a complex containing RNA and SFPQ directly interacts with a tetrameric kinesin complex containing the motor, KIF5A, and the adaptor, KLC1 , furtherCollectively, these data strongly indicate that SFPQ plays important roles in the nucleus and axon of neurons, and that the balance in the nucleocytoplasmic distribution of SFPQ is critical for the development and maintenance of neuronal integrity and function.Not surprisingly, due to the emerging physiological roles of SFPQ in neuronal development and homeostasis as described above, an increasing number of recent literature has demonstrated the implications of SFPQ in neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) frontotemporal lobar degeneration (FTLD), and Alzheimer’s disease (AD) ,62,63,64The cytoplasmic mislocalization and aggregation of RBPs have been emerging as new hallmarks of neurodegenerative diseases ,4,69. ThThe molecular mechanisms by which the nuclear pool of SFPQ is mislocalized to the cytoplasm and forms aggregates remain to be defined. However, several lines of evidence suggest that aberrant interactions of SFPQ with other misregulated or mutated proteins cause these pathological features. Interestingly, most of these proteins are neurodegenerative disease-implicated proteins such as tau, TDP-43, and FUS. Overexpression of pathological tau mutant (P301L) previously identified in familial cases of FTLD promotes cytoplasmic accumulation and aggregation of SFPQ . The famAltered zinc homeostasis has also been proposed as a potential mechanism of the cytoplasmic accumulation and aggregation of SFPQ . AlteredAlthough whether loss of the nuclear pool of SFPQ is a cause or merely an effect of the progression of the diseases is yet to be defined, given the predominant presence of SFPQ in the nucleus and the important nuclear roles of SFPQ in many aspects of RNA biogenesis and the maintenance of genome stability, imbalance in the nucleocytoplasmic distribution of SFPQ is likely to contribute to the onset and/or progression of the diseases by loss-of-function mechanisms . In addition, SFPQ was shown to bind extensively to its retained intron, which leads to nuclear loss and cytoplasmic accumulation of SFPQ. These data led to the proposal that intron retention of the sfpq transcript and nuclear loss of SFPQ are general molecular hallmarks of familial and sporadic ALS [sfpq transcript, which promotes the mislocalization of FUS to the cytoplasm [sfpq transcripts and the interaction of SFPQ and FUS with the retained intron was proposed as an underlying mechanism of mislocalization of SFPQ and FUS in ALS [Interestingly, aberrant RNA splicing of the d in ALS ,75. Abnoadic ALS . In a foS in ALS ,75.Stress granules are another type of membraneless organelles governed by liquid–liquid phase separation . Stress Many lines of evidence point to cytoplasmic accumulation of SFPQ as an emerging hallmark of neurodegenerative diseases. As mentioned previously, nucleus-to-cytoplasmic redistribution of SFPQ is most likely to cause loss of nuclear function. Whether the cytoplasmic aggregates of SFPQ found in the neurodegenerative diseases confer gain-of-function in the form of cytotoxicity is an intriguing possibility.Recent findings on the role of SFPQ and its implications in neurodegenerative diseases are reminiscent of TDP-43 in many ways. Both SFPQ and TDP-43 are RNA- and DNA-binding proteins and play multiple roles in RNA biogenesis and the maintenance of genome stability. A recent finding on the involvement of TDP-43 in DNA damage repair  further The role of SFPQ, in concert with FUS, in maintaining the isoform ratio of tau, a primary component of neurofibrillary tangles in the neurodegenerative diseases, further interweaves the intricate network of SFPQ with other neurodegenerative disease-linked RBPs. However, the precise molecular mechanisms that coordinate these complicated networks spatially and temporally are yet to be elucidated. Thus, further understanding of the precise mechanisms underpinning the interactions of SFPQ and other RBPs may also reveal the molecular mechanisms underlying the onset and progression of neurodegenerative diseases. Pathological protein aggregation manifested as neurofibrillary “tangles” is a hallmark of a number of neurodegenerative diseases including ALS and AD. SFPQ, as presented in this review, participates in the intricate network with many other known neurodegenerative disease-linked RBPs and tau. Given the complex regulatory roles of SFPQ through interactions with a wide array of interaction partners including DNA, RNA, and proteins, finding ways to “untangle” these interactions and navigate through the intricate regulatory network of SFPQ may present future diagnostic and/or therapeutic avenues for neurogenerative diseases.


Mycobacterium tuberculosis (Mtb) H37Rv accounts to more than 65 genes, 51 of which are thought to express a functional protein. PE_PGRS proteins share a conserved structural architecture with three main domains: the N-terminal PE domain; the PGRS domain, that can vary in sequence and size and is characterized by the presence of multiple GGA-GGX amino acid repeats; the highly conserved sequence containing the GRPLI motif that links the PE and PGRS domains; the unique C-terminus end that can vary in size from few to up to ≈ 300 amino acids. pe_pgrs genes emerged in slow-growing mycobacteria and expanded and diversified in MTBC and few other pathogenic mycobacteria. Interestingly, despite sequence homology and apparent redundancy, PE_PGRS proteins seem to have evolved a peculiar function. In this review, we summarize the actual knowledge on this elusive protein family in terms of evolution, structure, and function, focusing on the role of PE_PGRS in TB pathogenesis. We provide an original hypothesis on the role of the PE domain and propose a structural model for the polymorphic PGRS domain that might explain how so similar proteins can have different physiological functions.To the PE_PGRS protein subfamily belongs a group of surface-exposed mycobacterial antigens that in  Mycobacterium tuberculosis (Mtb) is one of the ancients and most successful human pathogens, still responsible for ≈ 10 million active TB cases and ≈ 1.5 million deaths in 2018 [Mtb infection is latent TB (≈ 95%), with no signs or symptoms of disease, characterized by a dynamic equilibrium between the host immune response and the bacillus which usually last for lifetime [Mtb interaction remain poorly understood as well as the bacterial proteins and virulence factors that provide Mtb with these unique features [Mtb belongs to the species Mycobacterium tuberculosis complex (MTBC), which is a monomorphic species subdivided in phylogeographic lineages that also include: M. africanum, that causes TB in humans but only in certain regions of Africa; M. microti, that causes TB in voles; M. bovis, that comprises several ecotypes that cause TB in wild and domesticated animals [Mtb, or rather MTBC, and other mycobacterial species (most importantly with Mtb progenitors as the smooth tubercle bacilli), indicated that the evolution of Mtb as a human pathogen has been mainly characterized by a process of gene loss and intragenomic recombination [lifetime . The immfeatures . Mtb bel animals . ComparaMtb are the pe and ppe genes, which evolved through multiple events of gene duplication, recombination and diversification, and occupy almost 10% of the Mtb genome coding capacity [Interestingly, among the gene families that are restricted and abundant in capacity –13. PE pcapacity .Mtb genome [Mtb genome contains 65 pe_pgrs genes, although only 51 of these express a functional protein, at least in Mtb H37Rv [M. marinum (≈ 148 genes) and M. ulcerans (≈ 121 genes), although pe_pgrs genes in these species show significant differences with those found in MTBC [The polymorphic GC-rich sequences (PGRS), which were first identified as repetitive genetic sequences and used as a typing tool in molecular epidemiology studies , were dib genome . The Mtbtb H37Rv . These garinum ≈ 8 genes a in MTBC . It is w in MTBC ,19. Herepe and ppe genes within the mycobacterial genus led to the identification of five coevolving gene subfamilies [Mycobacterium smegmatis and Mycobacterium abscessus, the closest to the genus common ancestor, possessing only few of these genes and some slow-growers species bearing tens of these genes [pe and ppe families appeared first associated with the esx-1 genetic locus coding the prototype of Type 7 Secretion Systems (T7SS) [esx-1 cluster followed by multiple duplication events of the pe and ppe genes led to the expansion of these families in the slow growing mycobacterial species  ,20. Dupl species . Pathogefamily V . Interesproteins ,21,22.Fipe_pgrs17/-18 [pe_pgrs3/-4, and pe_pgrs50/-51 clusters. Intragenic and intergenic recombination in pe_pgrs genes was associated with mutations and indels that led to the expansion and diversification of pe_pgrs genes. However, it is not clear how horizontal gene transfer processes might have contributed to the expansion and diversification of pe_pgrs genes in STB [M. marinum, as M. riyadhense, M. lacus, M. decipiens, and M. shinjukuense highlighted the presence of at least some pe_pgrs genes [pe_pgrs genes in these mycobacterial species belonging to the MTB-associated phylotype will shed new light on the role of these genes in the evolution of MTBC.Comparative genomic studies in MTBC and in smooth tubercle bacilli (STB) highlighted several examples of gene duplication events, including for instance the rs17/-18 , pe_pgrss in STB . The genrs genes ,25. Thesrs genes . A fine pe_pgrs genes that continued to evolve at a slower pace by intragenomic rearrangements, mutations, and indels  amino acids at position 7–8 . The PE-pe/ppe genes are evolutionary associated with the ESX gene locus and PE/PPE dimers are indeed structurally similar to the ESXA/B and homologous proteins, which are actively secreted antigens known for their immunogenicity [Most of the knowledge on the structure and function of the PE domain comes from studies on PE/PPE protein couplets ,35, whicgenicity ,37. The genicity ,22.Mtb allow for the determination of homology model structures. An example of homology model computed with MODELER software [pe_pgrs genes as single operons.Interestingly, despite a significant degree of amino acids sequence homology, the PE domain of different proteins within the PE/PPE and PE-unique subfamilies show a certain degree of variability, as observed by the poorly conserved hydrophilic/hydrophobic profiles of the amino acidic residues ,c. Convesoftware  using thsoftware ,39. It wsoftware . Recent software . We suggThe presence of multiple repeats containing the GGA-GGX motif interspersed with unique sequences is the feature of the PGRS domain . The PGRpe_pgrs genes are highly conserved and are subjected to purified selection in Mtb [II) [II, like polyproline type II-like (PPII), form flexible left-handed extended helices, which are not constrained by intra-helix hydrogen bonding as in the case of alpha helices. To gather information on the structural features of PGRS domains, homology modeling is extremely useful. Indeed, using the PGRS domain of PE_PGRS30 as a case study, consensus-based sequence alignment using HMMer identifies a structure . Using this alignment, a reliable homology model can be obtained with MODELER [II helices  . PGII, l MODELER , that is helices ,b. As inar prism   and unique amino acids sequence at the extreme protein C-terminal end. However, in nine PE_PGRSs  the unique C-terminal domain is larger and can reach ≈ 300 amino acids in length. Interestingly, PE_PGRS3 and PE_PGRS50 present a homologous, arginine-rich C-terminal domain. Similarly, the unique C-terminal domain of PE_PGRS30 is highly homologous to the C-terminal domain of the PE-unique protein encoded by Rv3812 . It is lactivity .The amino acid sequence that usually extends from position ≈ 90–92 to position 135–140 of the PE_PGRS proteins bridges the PE and PGRS domains . The proMtb involving PE_PGRSs have so far hampered the establishment of a working model that can identify the protein domains responsible for protein translocation and localization on the mycobacterial surface. Heterologous expression of PE_PGRSs in M. smegmatis demonstrated that PE_PGRS can reach the mycobacterial surface and that the PE/l-GRPLI domain is necessary and sufficient to warrant translocation of the whole protein on the mycobacterial surface despite the fact that the ESX-5 T7SS is missing [M. smegmatis. Indeed, expression in M. smegmatis or M. bovis BCG of a recombinant fusion protein expressing MPT64 downstream the PE/l-GRPLI domain of PE_PGRS33 resulted in the surface localization of MPT64, where it remained tightly associated with the mycobacterial cell wall [M. marinun and in Mtb showed that PE_PGRS proteins are secreted in an ESX5-dependent mechanism [M. marinum culture supernatant by using an anti-PGRS monoclonal antibody or by specifically detecting PE_PGRS45, and secretion was abolished in the ESX-5 mutant [ppe38/ppe71 region, or natural mutations in this gene locus occurring in some Mtb Beijing strains and in M. bovis BCG, affected PE_PGRSs secretion in MTBC providing compelling evidences for the ESX5-dependent secretion of these proteins [M. marinum LipY and PE_PGRS proteins are processed by PecA, a PE_PGRS protein itself, that cleaves the PE domain on the mycobacterial surface. Interestingly, PecA can cleave the Mtb LipY (LipYtub) protein when heterologously expressed in M. marinum, suggesting that the same processing may occur in Mtb. While these studies significantly contributed to better understanding the mechanisms of PE_PGRS protein translocation in Mtb and the consequences in TB pathogenesis and Mtb virulence, many aspects remain obscure. For instance, the fact that certain PE_PGRS proteins can be extracted by using non-anionic detergents as Genapol [M. marinum homologue of Mtb PE_PGRS35, could not be found in the culture supernatant despite being cleaved of the PE domain in the mycobacterial surface, pointing for an association with the mycobacterial outer membrane [The paucity of studies on  missing ,51,53. Iell wall . As expeell wall ,55. Howeechanism ,56,57. P5 mutant . More reproteins . In a reproteins  demonstr Genapol ,53 sugge Genapol . More remembrane .II-like helices, closely spaced together to form flat multiple-layer domains [II fibrils to interact with host components or other molecules, or inwards, to ensure proper embedding of the protein on the mycomembrane. Indeed, the presence of multiple hydrophobic phenylalanine-leucine/isoleucine residues in these intercalating sequences lend support to this hypothesis  and PE_PGRS4 , are two highly homologous PE_PGRSs. PE_PGRS3 presents a unique and peculiar arginine‐rich C‐terminal domain (≈ 80 aa) which is not present in PE_PGRS4. The highly homologous PGRS domain of the two proteins presents an extra GRPLI motif at position 528–532 and 421–425, for the PE_PGRS3 and PE_PGRS4, respectively.pe_pgrs [Mtb lineages [Mycobacterium canetti and Mycobacterium bovis, but not in Mtb strains, is somehow intriguing.The presence of these two neighboring, highly homologous genes is a classic example of a gene duplication event, which is a hallmark of other pe_pgrs ,60. Indepe_pgrs4 constitutively expressed and pe_pgrs3 specifically expressed following long-term exposure to low inorganic phosphate concentrations [M. smegmatis demonstrated that the arginine-rich domain is available on the mycobacterial surface, can significantly affect net surface charge and can promote adhesion to host cells and tissues [Mtb are required to better characterize the role of PE_PGRS3 in TB pathogenesis, it is tempting to speculate that the peculiar expression pattern and unique arginine-rich domain may directly implicate PE_PGRS3 during the persistence of Mtb in low phosphate environments as macrophages and granulomas.Interestingly, these two genes were shown to be differentially regulated, with trations , undersc tissues . While min silico analysis of the PE_PGRS5 predicted the presence of an extended disordered region within the PGRS domain containing seven endoplasmic reticulum signal peptides [Mtb residing in infected host cells, may lead to translocation of the protein in the endoplasmic reticulum and activation of specific host pathways. However, proper localization of PE_PGRS5 and other PE_PGRS proteins in macrophages or other Mtb infected host cells is required to support this hypothesis.PE_PGRS5 (Rv0297) is a 591 amino acids protein which shows a very high similarity with the N-terminal domain of PE_PGRS33, though a substantial variation is observed between the amino acid sequences in the PGRS domain. Furthermore, peptides . Indeed,peptides . Interespeptides . It can M. smegmatis enhanced resistance to H2O2-induced oxidative stresses in infected lung epithelial cells, which was dependent upon the enzymatic activity of the phosphoglycerate mutase domain. Interestingly, interaction of PE_PGRS11 with TLR2 triggered COX-2 and Bcl2 expression in infected cells, with these anti-apoptotic signals mediating resistance to oxidative stresses. Moreover, expression of the pe_pgrs11 gene was found to be up-regulated in hypoxic conditions, which are thought to occur within granulomas [Mtb surface and its expression profile, prompted to hypothesize that PE_PGRS11 can interact with host components and contribute to allow evasion of Mtb from oxidative stresses [PE_PGRS11 (Rv0754) is a 584 amino acids protein with a very short PGRS domain . Interestingly, the amino acid sequence that extends from the end of the PE domain to the GRPLI motif (the linker domain l) is longer than what observed in most PE_PGRSs and a Pro→Glu substitution is present in the GRPLI . The unianulomas ,64. The stresses . PE_PGRSstresses . While nPE_PGRS16 (Rv0977) is a 923 amino acids protein characterized by a large C-terminal domain of 273 amino acids downstream of the PGRS domain. Particularly, the unique C-terminus presents a marked hydrophobicity as assessed by ≈ 88% of not polar (45.5%) and polar, but not charged amino acid residues (42.8%). The structural characterization of the unique C-terminal domain demonstrated the presence of an aspartic proteinase-like domain . Despitepe_pgrs16 was upregulated under nutrient-depleted growth conditions, in bone marrow infected macrophages and finally in aerogenically infected mice [Intriguingly, ted mice . Althougpe_pgrs17 is a recombination hot-spot site [PE_PGRS17 (Rv0978c) and PE_PGRS18 (Rv0980c) are 331 and 457 amino acids proteins, respectively, with a large and unique C-terminal domain. The two genes share a high degree of homology, suggesting the occurrence of an intra-genomic duplication event . Indeed,pot site .Salmonella enterica serotype Typhi [M. smegmatis. PE_PGRS17 binds to TLR2 and activate the ERK1/2, p38 MAPK and NF-κB signaling pathway, promoting TNF-α secretion [M. smegmatis promoted cell death and enhanced intracellular mycobacterial survival over parental M. smegmatis strains, similarly to what observed for other PE_PGRSs. However, the role and contribution of the unique C-terminal domain in this process is still unclear.The unique C-terminal domains of these two PE_PGRSs share a very high homology (≈ 90%) and blast analysis reveals that this domain has a high similarity with the YncE family of proteins described to play a role in bacterial survival of pe Typhi . The onlecretion ,69. PE_Pecretion . Heterolpe_pgrs genes, transcriptional analysis of pe_pgrs26 indicates its downregulation in Mtb infecting macrophages or during the chronic-persistent phase of infection in mice [MtbΔpe_pgrs26 mutant showed an attenuated phenotype during the acute phase of infection, but virulence was rescued during chronic/persistent phase (70 day post-infection), suggesting that this protein may be necessary during the acute phase [PE_PGRS26 (Rv1441c) is 491 amino acids protein with the typical PGRS domain. Contrary to other  in mice . Accordite phase ,67.Mtb infection of macrophages, poly-ubiquitin chains bind to PE_PGRS29 available on the mycobacterial surface in an E3 ligases-independent manner and that Mtb ubiquitination occurred either in the permeable Mtb-containing phagosome or in the bacilli surviving in the cytosol. Interestingly, the MtbΔpe_pgrs29 mutant showed enhanced survival in infected macrophages compared to the parental Mtb strain, indicating that PE_PGRS29-dependent ubiquitin targeting of mycobacteria is crucial for the Mtb autophagic clearance. Moreover, lack of PE_PGRS29 abolished binding of particles containing the autophagosomal – associated protein LC3 and resulted in the accumulation of bacilli in the cytosol, suggesting that PE_PGRS29 is important for the autophagic clearance of cytosolic Mtb. In vivo experiments confirmed the enhanced virulence of the MtbΔpe_pgrs29 over the parental Mtb strain, with the former showing enhanced bacterial loads, inflammation and tissue damage. These results suggest that the PE_PGRS29-ubiquitin interaction mediating autophagic clearance of Mtb may be a smart strategy deployed by Mtb to achieve long-term intracellular survival in infected macrophages while avoiding excessive and potentially deleterious inflammatory responses.PE_PGRS29 (Rv1468) is a 370 amino acids protein with a short C-terminal domain (≈ 11 amino acidic residues) and the classical l-GRPLI motif with an unusual substitution of the glycine with the polar amino acid asparagine. Chai et al.  identifiM. marinum, which is specifically upregulated in granulomas [pe_pgrs30 gene is upregulated during growth in infected macrophages and in murine host tissues during the chronic persistent phase of infection [MtbΔpe_pgrs30 mutant shows an attenuated phenotype in infected mice, mainly during the chronic phase of infection, with a dramatic drop in bacilli in the lung tissue and a remarkable reduction in tissue damage compared to the parental strain [MtbΔpe_pgrs30 with a plasmid expressing a functional deletion mutant of PE_PGRS30 missing the C-terminal domain, fully restored the mutant virulence, pointing to the key role of the PGRS domain [in vitro experiments carried out in macrophages confirmed that PE_PGRS30 is required to block phagosome maturation by Mtb and again that the unique C-terminal domain is dispensable for this process [Mtb and as such can be considered a virulence factor, although the exact mechanism involved in this process remains to be determined. There are indications that the C-terminal domain of this protein is not available on the surface [Mtb pathogenesis.PE_PGRS30 Rv1651c) is a 1011 amino acid protein which presents a unique and large C-terminal domain of 306 amino acids. Characteristically, PE_PGRS30 shows a high homology with the MAG24 protein of anulomas . Furtheranulomas , a PE-un51c is a nfection . In linel strain . InteresS domain . Moreove process . Hence,  surface  and may pe_pgrs33 gene is constitutively expressed, with the level of transcripts detectable and similar in axenic culture, during infection of macrophages and in infected host tissues [Mtb infection, although the multiple repeats and redundancy of the PGRS sequence, which is the domain recognized by the host antibodies, makes it difficult to assess the specificity of this response [PE_PGRS33 (Rv1818c) is a 498 amino acids protein and it is the first and probably the most studied protein of the family. It is a classical PE_PGRS protein with a short (≈ 10 amino acids) unique C-terminal domain. Several studies indicated that the  tissues ,73–75. Sresponse ,76,77.M. smegmatis promoted cell death and increased mycobacterial survival in macrophages and in intraperitoneally infected mice over the parental strain or the M. smegmatis recombinant strain expressing only the PE domain of PE_PGRS33, pointing for the key role of the PGRS domain in this process [M. smegmatis expressing PE_PGRS33 compared to controls [M. smegmatis expressing PE_PGRS33, further supporting the key role of PE_PGRS33-TLR2 interaction [MtbΔpe_pgrs33 mutant was impaired, compared to the parental strain, in its ability to enter in macrophages, but not epithelial cells, in a process involving activation of the TLR2-CR3 pathway, which activates the inside-out-signaling to promote Mtb entry in macrophages [MtbΔpe_pgrs33 complemented with a series of functional deletion mutants missing different portions of the PGRS domain suggest that the proximal PGRS domain (amino acid sequence encompassing positions 140–260) is sufficient to activate TLR2 [Heterologous expression of PE_PGRS33 in  process ,78,79. Pcontrols ,78,80,81controls ,79. Of ncontrols  and propcontrols . Moreoverophages . Interesate TLR2 .in vivo experiments in mice, which rather than showing attenuation of the mutant compared to the parental strain showed an enhanced virulence during the chronic steps of the infection [MtbΔpe_pgrs33 complemented with a naturally occurring frameshifted pe_pgrs33 allele, obtained from an Mtb strain belonging to an ancient lineage, caused enhanced tissue damage during the chronic steps of infection [pe_pgrs33 in a collection of Mtb strains representative of the different phylogeographic lineages highlighted that this gene was under purifying selection, confirming other findings on other pe_pgrs genes [pe_pgrs33 polymorphisms in Mtb clinical isolates, showing that large in-frame indels and frameshift mutations correlated with the absence of cavitation in lungs [pe_pgrs33 do not affect Mtb virulence [pe_pgrs33 is missing in M. marinum and smooth tubercle bacilli [Mtb suggests that MTBC acquired pe_pgrs33 during the evolution to promote tissue damage and persistence in the lung tissue. Indeed, we hypothesize that it may play a critical role in the successful transmission of Mtb in humans [The role of PE_PGRS33 in TB pathogenesis has been further investigated in nfection . Similarnfection . Geneticrs genes . Previouin lungs  or with in lungs . Since tirulence . The fin bacilli  and it in humans .M. marinum homolog of the PE_PGRS35 (MMAR_2933) can process the Mtb protein LipY (LipYtub) when expressed as heterologous protein in M. marinum. Processing of LipYtub by the M. marinum PE_PGRS35 homologue occurs at multiple sites, near or within the YxxxD/E secretion motif within the PE domain or in the linker domain of LipY, in line with previous findings [M. marinum and cleavage occurs at the mycobacterial cell surface where the protease can cleave itself and other PE_PGRS proteins. It remains to be determined the role of PecA (PE_PGRS35) in Mtb.PE_PGRS35 (Rv1983) is a 558 amino acids protein that contains a large unique C-terminal domain that shows 43,9% identity with the C-terminal domain of the PE_PGRS16 and comprises an aspartic proteinase domain. In a very recent report, Burggraaf et al.  showed tfindings . Since tfindings . Interespe_pgrs41 belongs to the aprABC gene locus that includes the two upstream genes (aprA and aprB), with aprC corresponding to pe_pgrs41. The two-component regulator phoPR senses the acidic pH of the phagosome and induces expression of the aprABC genes [aprABC, which is unique to MTBC, in the mechanism that grants adaptation of Mtb to intracellular lifestyle. However, expression levels of the aprC/pe_pgrs41 genes are much lower and poorly modulated compared to what observed for the aprAB genes. Moreover, the functional relation between the proteins encoded by the aprABC locus remains undisclosed. In line with these findings, heterologous expression of PE_PGRS41 in M. smegmatis enhanced mycobacterial survival in macrophages thanks to the inhibition of autophagy, increased cytotoxicity, and dampening of inflammatory responses [PE_PGRS41 (Rv2396) is a small PE_PGRS protein of 361 amino acids. Like PE_PGRS11, PE_PGRS41 has a longer l-GRPLI domain compared to most PE_PGRSs. Interestingly, BC genes . These responses . Hence, esponses .Mtb pathogenesis. The MtbΔpe_pgrs47 mutant is impaired in its ability to replicate intracellularly in macrophages or to persist in host tissues at late stages of Mtb infection in mice [MtbΔpe_pgrs30 mutant [Mtb antigens as TB9.8 and Ag85B at the early and chronic-persistent phase of infection [Mtb infected macrophages provides a potential mechanism for the observed inhibition of antigen processing and presentation [PE_PGRS47 (Rv2741) is a 525 amino acid protein that recent studies implicated in  in mice , similar0 mutant . Interesnfection . Interesentation . These rentation ,12.Mtb biology. Studies that assessed the transcriptional expression profile of pe_pgrs genes demonstrated that some genes were specifically expressed or up-regulated under certain environmental conditions  or in Mtb residing inside macrophages, and that level of expression of different pe_pgrs genes could vary significantly [Mtb constitutively expresses pe_pgrs33 while pe_pgrs30 transcription is specifically activated in Mtb-infected macrophages [pe_pgrs genes are scattered in the genome where they are organized in single open reading frames, similarly to ppe_mptr genes [pe/ppe couplets, whose genes are co-expressed and the corresponding proteins form dimers and are functionally linked [pe_pgrs genes are in purifying selection in Mtb [Mtb biology, rather suggesting that each protein may exert a unique function.Despite the relative high homology in amino acid sequence, structural organization, and the repetitive and apparently redundant PGRS domain, the experimental evidences collected so far indicate that different PE_PGRS proteins can play distinct roles in ficantly ,61,67. Frophages ,50. Thestr genes , but dify linked ,33,88. TMtb [Since the identification of PE_PGRS proteins in Mtb  several Mtb entry in host cells: PE_PGRS3, by promoting adhesion to macrophages and epithelial cells through the “sticky” arginine-rich C-terminal domain [Mtb entry in host cells.PE_PGRS3 and PE_PGRS33 promote l domain ; PE_PGRSl domain , which wl domain . It remaMtb deploys a sophisticated arsenal of protein and effector molecules to prevent phagosome-lysosome fusion and evade killing [Mtb are present in five copies [Mycobacterium bovis Bacille Calmette and Guerin (BCG) is the main cause of attenuation [Mtb survival and replication in macrophages requires secretion through the ESX-1 system of ESXA/B and other proteins that permeabilize the phagosome membranes to promote access of Mtb proteins to the cytosol and prompt bacilli translocation in the cytoplasm [Mtb intracellularly and prevents phagosome acidification in a yet unknown mechanisms, thereby enhancing the survival of Mtb in macrophages [Mtb survival and virulence in macrophages, others PE_PGRSs seem to counterbalance these processes by modulating autophagy. Autophagy can support bacterial clearance in macrophages, either by engulfing phagosomes-containing bacteria (xenophagy) or by targeting cytosolic bacteria with ubiquitin and LC3 [Mtb, or bacilli otherwise accessible to ubiquitin in the permeabilized phagosome, to trigger host xenophagy and promote bacterial clearance, apparently to reduce inflammatory responses [Mtb infected macrophages with important consequences not only in bacterial survival but also in antigen presentation of key Mtb antigens [M. smegmatis [Mtb intracellular survival, antigen presentation and associated inflammatory responses.Following entry in macrophages and engulfment in phagosomes,  killing . Among te copies . ESX-1 ienuation . Mtb surytoplasm ,94. Amonrophages ,95. PE_Prophages . While s and LC3 . PE_PGRSesponses . Conversantigens  and PE_Pmegmatis . The curThe ability to promote inflammatory responses, oxidative stresses and cell death primarily by cell necrosis has been demonstrated for PE_PGRS11, −17, −18, −30 and −33. Usually, PE_PGRSs can trigger the secretion of inflammatory cytokines as TNF-α and IL-12 by binding to TLR2 and activation of the downstream signaling cascade ,55,78–81in vivo models of infection and the few results available highlight a complex picture that again prevents the disclosure of a common and overarching mechanism for the role of PE_PGRS in TB pathogenesis. PE_PGRS proteins are among the most abundant Mtb proteins in granulomas during acute and chronic TB disease in guinea pigs [pe_pgrs genes are differentially regulated in host tissues during Mtb infection [pe_pgrs genes resulted in an attenuated phenotype in vivo when immunocompetent mice were infected as for the MtbΔpe_pgrs30 [MtbΔpe_pgrs47 [MtbΔpe_pgrs33 [MtbΔpe_pgrs29 [pe_pgrs genes may lead to different and apparently conflicting results, a key and common feature of these in vivo experiments is the emergence of a clear phenotype in the Mtb mutant during the chronic persistent steps of infection (Mtb pe_pgrs mutants in animal models of infection that better mimic human TB, as aerogenic infection of guinea pigs, rabbits, or non-human primate.The consequences of these events taken place at cellular level have been investigated in nea pigs , and pe_nfection ,80. Inace_pgrs30  and MtbΔe_pgrs47  mutants,e_pgrs33  and the e_pgrs29  mutants nfection . Interesppe38/ppe71 gene locus abolished secretion of PE_PGRS and PPE_MPTR proteins in Mtb, resulting in an Mtb strain with enhanced virulence, at least in mice [MtbΔppe38/ppe71 mutant and parental Mtb CDC1551 strain diverged primarily during the chronic/persistent steps of the infectious process in mice, with a significant impact on lung bacterial burden and most importantly tissue damage in the lung parenchyma [Mtb pathogenesis. The impact that genetic polymorphisms of the pe_pgrs33 have on the clinical outcome of TB disease [Mtb virulence as assessed in the experimental mouse model of TB, suggest that at least some PE_PGRS proteins can exert important immunomodulatory activities in TB pathogenesis. It has been hypothesized that the strong selective pressure on pe_pgrs33 combined with its role in TB pathogenesis indicate its involvement in TB transmission [Mtb virulence in the host tissue to permit prolonged survival in the infected host [In line with these findings, inactivation of the  in mice . Interesrenchyma . These f disease ,98 and osmission . Moreoveted host .Mtb one of the most successful human pathogens. We suggest their involvement primarily in supporting the ability of the tubercle bacilli to survive in the presence of a strong immune response and in modulating the complex inflammatory processes that shape the dynamic host–microbe interaction that can lead to active disease, primarily in the lung tissue, where the extensive tissue damage is instrumental for Mtb transmission (Although more experimental evidences are needed to elucidate the role of PE_PGRS proteins in TB pathogenesis, the current body of evidences suggest that these proteins may have emerged and evolved in MTBC to resist, modulate or manipulate the complex mammal host immune system. In line with this hypothesis, PE_PGRSs are expected to contribute significantly to the main abilities that make smission .Mtb as a human pathogen, whose survival is dependent upon the ability of the bacilli to spread from a patient with active pulmonary TB to a naïve host, was accompanied by significant genetic changes compared to the environmental mycobacterial progenitors. Among the most relevant are the switch of Mtb into a monomorphic bacterium, the marked reduction in genome size accompanied by the expansion and diversification of genes belonging to the pe and ppe families. The set of pe_pgrs genes in Mtb are overall genetically well conserved and in positive selection [Mtb biology different from that proposed earlier which implicated PE_PGRS proteins in antigenic variability as a source for immune evasion strategies [II sandwich modules. These PGII units are instrumental for the PGRS anchoring to the mycomembrane outer leaflet while allowing proper exposure of unique amino acids found on the opposite side of the sandwich outward, where they are available for interaction with host components. From this perspective, the variable sequences present in between GGA-GGX conserved spacers would contribute either to the anchoring of the protein to the cell or to its specific function, depending on the side of the sandwich in which they are exposed. The large variability of these variable sequences among PE_PGRS proteins might help to explain why proteins with so similar structural features have so different roles in Mtb physiology.The emergence of election , suggestrategies . In thisClick here for additional data file.


We proposed a two-step poling technique to fabricate nanoscale domains based on the anti-parallel polarization reversal effect in lithium niobate on insulator (LNOI). The anti-parallel polarization reversal is observed when lithium niobate thin film in LNOI is poled by applying a high voltage pulse through the conductive probe tip of atomic force microscope, which generates a donut-shaped domain structure with its domain polarization at the center being anti-parallel to the poling field. The donut-shaped domain is unstable and decays with a time scale of hours. With the two-step poling technique, the polarization of the donut-shaped domain can be reversed entirely, producing a stable dot domain with a size of tens of nanometers. Dot domains with diameter of the order of ∼30 nm were fabricated through the two-step poling technique. The results may be beneficial to domain-based applications such as ferroelectric domain memory. Lithium niobate (LiNbOro-optic ,2, acousro-optic ,5,6,7 prro-optic  and nonvro-optic ,10. Recero-optic ,12, has ro-optic ,14,15,16Recently, various novel LNOI-based optical elements have been reported, such as electro-optic modulator , high-Q However, several groups have reported an anti-parallel polarization reversal or an anomalous domain inversion effect in various ferroelectric thin films, including BaTiO3 , PZT [243 ,26, LiTa3 [3 ,28, and 3 [3 [3 ,30,31,323 [3 [3 ,34,35,36f of the pt-coated Si probe was 100 kHz, respectively.A decreases with the increase of both UFor a dot domain written by the AFM tip, its size increases with the magnitude U voltage ,16,37. Teviously ,25,31,32o et al. . Surely,We demonstrated a simple two-step poling technique to eliminate the inner domain while keeping the shape and size of the whole domain unchanged. After the above-described step-1 poling process, a donut-shaped domain was generated, then one applied another poling field on the AFM tip exactly at the same position as that in the step-1 poling stage but with opposite field polarity, this was the step-2 poling stage. With an appropriate magnitude UWe also studied the effect of magnitude and pulse duration of the step-2 poling field on the reversal efficiency of the inner circular domain. In the experiments, firstly we produced a set of donut-shaped domains with a step-1 poling field of UA. This was confirmed experimentally, as shown in It was reported that the domain poling in LiNbOd poling ,41. Thisd poling . Also, cd poling ,31. ThesIn summary, we demonstrated that a donut-shaped domain would be generated due to the anti-parallel polarization reversal effect in LNOI under the action of an AFM tip poling field. We proposed a two-step poling technique to reverse the polarization of the inner domain of the donut-shaped domain. Furthermore, it is possible to fabricate a stable circular domain with a size of tens of nanometers through this two-step poling technique based on the anti-parallel polarization reversal effect, and dot domain with diameter ∼30 nm was produced. This technique may be beneficial to domain-based applications such as ferroelectric domain memory.


The effect of equal channel angular pressing (ECAP) through the route Bc and aging treatment on the grain structure and properties of the Cu–1Cr–0.2Si alloy was investigated. Microstructure was detected by scanning electron microscopy (SEM), x-ray diffraction (XRD), and electron backscatter diffraction (EBSD) and the mechanical properties and electrical conductivity were tested. Results shown that after ECAP, accompanying the grains refined to nano-and submicron-structure, the Cr particles were gradually spread along the grain boundaries (GBs), aging treatment promoted Cr particles dispersed in the matrix. ECAP greatly increased the ultimate tensile strength (UTS) while having a small effect on the conductivity, and aging treatment increased electrical conductivity. The stable {111}<110> texture after ECAP and the lower dislocation density after aging treatment maybe the main reasons for the high conductivity of the material. Theuctivity ,8.Severe plastic deformation (SPD) can effectively improve the microstructure of metal materials such as Al alloys, Cu alloys, Mg alloys, etc., and greatly improve the uniformity of microstructure , howeverXu et al.  prepared3: 5 g, HCl: 50 ml, H2O: 100 mL). Microstructures were performed by a Quanta FEG-450 scanning electron microscope  equipped with INCAEDS software. A D8ADVANCE x-ray diffractometer  was used to characterize the macroscopic orientation. Using a WDW-300D microcomputer  controlled electronic universal testing machine to complete the tensile tests to measure the tensile strength with an error range of ±0.5%. The conductivity was tested by a Sigma2008B/C digital eddy current metal conductivity meter  with an error range of 0.1%.Cu–1Cr–0.2Si  alloy was prepared by high-purity electrolytic copper Cu (99.9%), Cu–1.2Cr , and a Cu–0.5Si  intermediate alloy. The ingot was homogenized at 900 °C for 5 h, and then extruded into a rod 30 mm in diameter. After multiple passes of cold rolling and drawing into a rod 12 mm in diameter, the alloy rod was then processed into Φ12 mm × 70 mm. ECAP deformation was conducted at room temperature using a die  up to eight passes through route Bc (where the sample is rotated 90° in the same direction between two consecutive passes) at the rate of 10 mm/s. The specimens were sampled along the extrusion direction and then aged at 350 °C for 1 to 8 h, respectively. All specimens were polished and corroded at room temperature for 10 seconds , after one pass, the orientation segregated in the {110} plane and the pole density reached nine. After two passes of deformation and aging, the strongest orientation rotated about 45° counterclockwise around the extrusion direction, accompanied by strain increase, and the base texture deflected and the density fluctuated, which indicated that strong interactions occurred in the microstructure during deformation. When the strain was lower, the orientation tended to the same crystal plane, which was consistent with the previous structure. The occurrence of dispersion indicated that under high strain, the original coarse grains were gradually refined after being broken, the microstructure tended to be uniformed, and some subgrains were transformed into grains with high angle grain boundaries (HAGBs).According to With the local recovery of the deformed structure, the aging treatment accelerates the precipitation process, which further causes the orientation to diverge and the texture strength to gradually decrease. For the different shear plane of adjacent passes by route Bc, the original texture had an effect on the texture that was produced by the first two passes of ECAP deformation . After fL. The local orientation difference reflected the orientation change in the plastic deformed zone. Although the dislocation density is not easy to measure directly, the relative size of dislocation density can be measured by the orientation change ,33. TherL and the dislocation density ρ at this point, which can be expressed by Kubin and Mortensen [L is the average value of the local orientation differences that can be obtained from the EBSD data.There is a quantitative conversion relationship between the local orientation difference θortensen ,35, as f14, ρ2 = 2.55 × 1014, ρ4 = 2.16 × 1014, and ρ8 = 1.87 × 1014. The change trend is shown in The dislocation density of each deformation pass was calculated successively as ρ1 = 2.41 × 10During ECAP deformation through the route Bc, the microstructure of the Cu–1Cr–0.2Si alloy was gradually refined into a fibrous structure, and then distributed along the extrusion direction. The high-density dislocations introduced by the deformation and lots of unstable subgrain boundaries and GBs caused by deformation lead to a continuous increase in lattice distortion energy, leading to an increase in the resistance of the free electrons to move, as shown in Previous studies have found that deformation and aging treatment directly affect the electrical conductivity of materials . In the (1) A high strength, high electrical conductivity, and high elongation Cu–1Cr–0.2Si alloy was fabricated by ECAP through route Bc + aging treatment ), and the strength of the alloy is expected to be further improve. (2) With an increase in the ECAP deformation passes, the tensile strength of the Cu–1Cr–0.2Si alloy was continuously increased. After four deformation passes, it reached 564 MPa, which was 42% higher than the undeformed material.(3) Aging treatment can promote the dispersion of Cr phases in the matrix, and greatly increase the conductivity of the Cu–1Cr–0.2Si alloy. With an increase in aging time, the quantity of the phases increased gradually and was uniformly dispersed in the matrix.(4) The strong {111}<110> and {111}<112> deformed textures and weak {001}<100> recrystallized textures were formed after eight passes of deformation. ECAP deformation and aging treatment can greatly increase the strength and conductivity of the material. The stable {111}<110> texture after ECAP and the lower dislocation density after aging treatment are the main reasons for the high conductivity of the material.


Drosophila photoreceptors, the phosphatidylinositol transfer protein (PITP) RDGB, is required for lipid transfer at endoplasmic reticulum (ER)–plasma membrane (PM) contact sites (MCS). Depletion of RDGB or its mis-localization away from the ER–PM MCS results in multiple defects in photoreceptor function. Previously, the interaction between the FFAT motif of RDGB and the integral ER protein dVAP-A was shown to be essential for accurate localization to ER–PM MCS. Here, we report that the FFAT/dVAP-A interaction alone is insufficient to localize RDGB accurately; this also requires the function of the C-terminal domains, DDHD and LNS2. Mutations in each of these domains results in mis-localization of RDGB leading to loss of function. While the LNS2 domain is necessary, it is not sufficient for the correct localization of RDGB, which also requires the C-terminal DDHD domain. The function of the DDHD domain is mediated through an intramolecular interaction with the LNS2 domain. Thus, interactions between the additional domains in a multi-domain PITP together lead to accurate localization at the MCS and signalling function.During phospholipase C-β (PLC-β) signalling in This article has an associated First Person interview with the first author of the paper. Summary: This study demonstrates interdomain interactions within a phosphatidylinositol transfer protein that regulate its localization and function at an ER-PM membrane contact site. The close approximation of intracellular membranes without fusion between them is emerging as a theme in cell biology . Such apin vivo function of several others is not clear. One group of LTPs named phosphatidylinositol transfer proteins (PITPs) mediate the specific transfer of PI between compartments. The first PITP identified and cloned was a protein with a single phosphatidylinositol transfer domain (PITPd) , phosphatidylinositol (PI), phosphatidic acid (PA), cholesterol and phosphatidylinositol 4-phosphate (PI4P) . These t (PITPd) . Since t (PITPd) ]. ImportDrosophila photoreceptors have emerged as an influential model system for the analysis of ER-PM contact sites  that has a well-annotated PITPd (RDGBPITPd). Loss of function or hypomorphic mutants for rdgB represented by 2rdgB and 9rdgB alleles respectively, show defective electrical responses to light, retinal degeneration and defects in light activated PIP2 turnover. RDGBPITPd has been shown to bind and transfer PI and PA in vitro, and is sufficient to support aspects of RDGB function in vivo , and expressed it in 9rdgB photoreceptors . We determined the localization of this protein by immunostaining with an antibody raised to the PITPd. Unlike full length RDGB which localized at the ER-PM junction, RDGBPITPd-FFAT was found to be mis-localized from the base of the rhabdomere . Collectively, these results imply that the domains present C-terminus to the FFAT motif contribute to localizing RDGB correctly which then impact its function.RDGB is essential to support the levels of PIPjunction . We test the eye . As prevransgene . When tecontrols D,E. The (DDHD-LNS2)Δ) and expressed this protein in 9rdgB photoreceptors  . Immunolocalization experiments revealed that RDGB(DDHD-LNS2)Δ was not localized to the ER-PM contact site but was distributed throughout the cell body Δ was unable to rescue the ERG phenotype of 9rdgB  and the PIP2 levels in 9rdgB flies expressing RDGB(DDHD-LNS2)Δ were comparable to that of 9rdgB flies , although probe levels were found to be unaltered across all genotypes . These findings imply that the presence of one or both of these domains is essential for correct localization and function of RDGB.There are two well annotated domains C-terminal to the FFAT motif in RDGB: DDHD and LNS2. Of these, the LNS2 domain has previously been implicated in the membrane localization of Nir2, the mammalian orthologue of RDGB ; 2015. Tell body A. An impLNS2Δ) and expressed the rest of the RDGB protein in photoreceptors of 9rdgB flies  . RDGBLNS2Δ was found to be completely mis-localized from the base of the rhabdomere, suggesting that this domain is indispensable for localization of RDGB . Similarly, PM PIP2 levels in 9rdgB reconstituted with RDGBLNS2Δ  was found to be as low as in 9rdgB photoreceptors  although probe levels were equal across all genotypes . These results collectively support an indispensable role for the LNS2 domain in supporting RDGB localization and function in vivo.Since our data shows that loss of both domains together lead to complete loss of RDGB function we then went onto investigate the role played by each of these individual domains. Firstly, to test if the LNS2 domain in RDGB is required for localization, we deleted the LNS2 domain , it localizes exclusively to the apical PM and not the basolateral PM in polarized photoreceptor cells implying underlying mechanisms which allow this preferential binding.While our sub-cellular fractionation assay reveals that the LNS2 domain is essential for membrane association of RDGB, it does not identify the cellular membrane to which the domain is targeted. To understand this, we cloned the LNS2 domain alone, tagged to GFP (LNS2::GFP) and expressed it in S2R+ cells. Under these conditions, LNS2::GFP was found to localize primarily to the PM with some punctate structures within the cell C,D,E. ToDrosophila photoreceptors, then what is the function of the DDHD domain, present just N-terminal to the LNS2 domain? To determine if this domain is essential for the function of RDGB, we at first checked whether the residues that give the domain its identity and nomenclature are present in RDGB. For this, we aligned the DDHD domain of PA-PLA1 with that of RDGB and determined that all four residues D, D, H and D are indeed also conserved in RDGB (DDHD/4A) in the full-length protein, expressed it in fly photoreceptors  and checked for its localization. RDGBDDHD/4A was found to be diffusely distributed and not localized to the base of the rhabdomere  and the LNS2 domain with the apical PM at the ER-PM MCS of  in RDGB A. To cheabdomere B. Thus t9rdgB reconstituted with RDGBDDHD/4A . Similarly, we found that the PIP2 levels in 9rdgB photoreceptors reconstituted with RDGBDDHD/4A were only modestly rescued compared to 9rdgB . These results collectively suggest that the DDHD domain is required for the correct localization and normal function of RDGB.Since altered localization leads to defects in RDGB function, we then tested if mutation of these conserved residues in the full-length protein also had a similar impact. We noted partial retinal degeneration in BDDHD/4A C and theBDDHD/4A D,E. Impoto rdgB9 F,G, althin vivo data shows that mutations in the conserved residues of the DDHD domain impact localization and function of the full length protein. To understand the function of the DDHD domain as a whole, we expressed an mCherry tagged version of the DDHD domain in S2R+ cells. We found that DDHD domain showed a diffuse distribution in the majority of cells, while in some cells a few punctate structures were also observed  in S2R+ cells along with GFP tagged LNS2 domain (LNS2::GFP). When we immunoprecipitated the DDHD domain using an mCherry antibody, we could detect the LNS2 domain in the pulled down fraction implying physical interaction between these two domains G,H.Drosophila RDGB, a multidomain PITP, it has been noted that re-expression of just RDGBPITPd which performs lipid transfer in vitro, in a null mutant background, is sufficient to rescue key phenotypes in vivo suggesting the sufficiency of the RDGBPITPd in supporting RDGB function. A more recent study has however shown that while RDGBPITPd can rescue key phenotypes, it is incapable of supporting lipid turn over during high rates of PLC-β signalling  suggesting a unique apical domain interaction partner that localizes it here. Studies on Nir2 have suggested the LNS2 domain binds PA , this lipid is not unique to or enriched at the apical PM. We also tested the role of the specific glutamic acid (D1128 in Nir2) within the LNS2 domain that has been reported to be required for PA binding and localization of Nir2 . Surprisingly, we found that a D1164A mutant of the LNS2 domain of RDGB was able to bind PA in vitro, just as well as wild type . When this mutation was introduced into the full length RDGB and expressed in 9rdgB photoreceptors , the mutant protein was still able to localize to the MCS similar to the wild type  and the basal PIP2 levels were also found to be unaffected .Thus the signal through which the LNS2 domain interacts specifically with the apical PM remains to be determined.How is RDGB accurately localized such that it can be concentrated at the ER-PM MCS? It has previously been demonstrated  that an o the PM , 2015, obinds PA ; while w of Nir2 . The equ2 levels on rescuing 9rdgB with RDGBDDHD/4A; analysis of a full deletion of the DDHD domain will be informative in establishing the full impact of loss of function in this domain on the activity of the RDGB protein. Surprisingly, and in sharp contrast to the LNS2 domain, when expressed by itself, the DDHD domain did not localize to the PM but showed a diffuse cytosolic distribution  was subcloned into pUAST-attB by using the restriction enzymes NotI and XbaI (NEB). Similarly, for making RDGB(DDHD-LNS2)Δ the cDNA corresponding to amino acids 1-655 was amplified, and for RDGBLNS2Δ the cDNA corresponding to amino acids 1–1000 was amplified and then individually subcloned in NotI and XbaI digested pUAST-attB. For cloning of rdgBDDHD/4A, mutations were introduced in the rdgB cDNA corresponding to amino acid numbers 776, 872, 894 and 902. Similarly, for cloning D1164ArdgB, mutation was introduced in the rdgB cDNA corresponding to amino acid number 1164. The resulting mutant genes were then subcloned in NotI and XbaI digested pUAST-attB. To clone the LNS2 domain alone, the cDNA of RDGB corresponding to amino acids 947-1259 was subcloned in pJFRC::GFP vector using the restriction enzymes BglII and NotI (NEB). A flexible linker of Gly(G)-Ser(S) of the sequence G-G-S-G-G-G-S-G-G-G-S-G-G was introduced between the LNS2 domain and GFP to allow independent and efficient folding of the two proteins. For cloning of the DDHD domain, the cDNA of RDGB corresponding to amino acids 730–913 was subcloned in BglII and XhoI digested pUAST-attB-mCherry with the flexible linker sequence present between mCherry and the DDHD domain. The DDHD-LNS2 construct was cloned by amplifying the cDNA corresponding to the amino acids 730–1259 of RDGB and tagging it to mCherry in BglII and XhoI digested pUAST-attB-mCherry, with the flexible linker sequence present between the mCherry and the DDHD domain.BDGP gold clone 09970 containing the S2R+ cells were cultured in Schneider's insect medium (HiMedia) supplemented with 10% Fetal Bovine Serum and with antibiotics Penicillin and Streptomycin. Cells were transfected using Effectene (Qiagen) as per manufacturer's protocol. Post 24 h of transfection, cells were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) and imaged to observe for GFP or mCherry fluorescence using a 60X 1.4 NA objective, in Olympus FV 3000 microscope.Heads of one day old flies were homogenised in 2X Laemmli sample buffer, and boiled at 95°C for 5 min. The samples were then run on a SDS-PAGE gel, and transferred on to a nitrocellulose membrane , with the help of a semi-dry transfer apparatus . The membrane was then blocked using 5% Blotto  in Phosphate-buffered saline (PBS) with 0.1% Tween 20 (Sigma-Aldrich) (PBST) for 2 h at room temperature (RT). The membrane was then incubated with the respective primary antibody, overnight at 4°C, using the appropriate dilutions . Following this, the membrane was washed in PBST thrice, and incubated with the appropriate secondary antibody  coupled to horseradish peroxidase, at RT for 2 h. The blots were visualized using ECL , and imaged in a LAS4000 instrument.For immunohistochemistry, retinae of one-day old flies were dissected under bright light in PBS. The samples were then fixed using 4% paraformaldehyde (Electron Microscopy Sciences) in PBS with 1 mg/ml saponin (Sigma Aldrich) for 30 min at RT. Post fixation, samples were washed thrice with PBS having 0.3% Triton X-100 (PBTX) and blocked using 5% Fetal Bovine Serum (ThermoFisher Scientific) in PBTX for 2 h at RT. The samples were then incubated overnight with the appropriate antibody in blocking solution at 4°C . Samples were then washed thrice with PBTX and incubated with the secondary antibody  at 1:300 dilution for 4 h at RT. For staining of the F-actin, Alexa Fluor 568–Phalloidin  at 1:200 dilution was added during incubation with the secondary antibody. Samples were then washed in PBTX thrice and mounted with 70% glycerol in PBS. For assessing retinal degeneration, retinae of 4-day-old flies were dissected and fixed as mentioned above, and incubated with Alexa Fluor 488–Phalloidin  at 1:200 dilution for 4 h at RT. The whole-mounted preparations were imaged under 60X 1.4 NA objective, in Olympus FV 3000 microscope.S2R+ cells were co-transfected with mCherry::DDHD and LNS2::GFP for 48 h, and lysed in ice-cold Protein Lysis Buffer . 10% of the lysate was aliquoted to be used as input. The remaining lysate was split into two equal parts. To one part, anti-mCherry antibody (ThermoFisher Scientific PA5-34974), (1.6 ug) was added, and to the other part, a corresponding amount of control IgG  was added, and incubated overnight at 4°C. On the next day, Protein-G sepharose beads  were spun at 13000X g for 1 min, and then washed with Tris-buffered saline (TBS), twice. The beads were then incubated with 5% Bovine Serum Albumin (BSA) (HiMedia) in TBS with 0.1%Tween-20 (TBST) for 2 h at 4°C. Equal amounts of blocked beads were then added to each sample, and incubated at 4°C for another 2 h. The immunoprecipitates were then washed twice with TBST containing β-Mercaptoethanol, and 0.1 M EGTA for 5 min. The supernatant was then removed, and the beads were boiled in 2X Laemmli sample buffer for western blotting.Drosophila heads were homogenised in ice-cold homogenisation buffer A . 10% of homogenate, representing the total head lysate, was directly taken for western blotting. The remaining homogenate was centrifuged at 5000 rpm for 5 min at 4°C to remove all chitinous material. The pellet was re-homogenized in the buffer to redeem any remaining membranous component from the cell ghost. This was done twice, post which the homogenate was spun at 100,000X g for 30 min, at 4°C to separate the entire membranous component from the cytosolic fraction. The pellet was reconstituted in buffer A. The re-suspended pellet representing the membrane fraction, and the supernatant representing the cytosolic fraction, were then individually used for doing western blotting.The assay was performed as described by S2R+ cells were individually transfected with pUAST-attB-mCherry::Spo20, pJFRC-LNS2(RDGB)::GFP and pJFRC-LNS2(RDGB-D1164A)::GFP for 48 h, following which cells were lysed with Protein Lysis Buffer . In parallel, strips made using nitrocellulose membranes  were spotted with increasing picomoles of DOPA . The spotted membrane was dried and then blocked using 5% BSA (HiMedia) in TBST for 2 h at RT. Following this, the strips were incubated overnight at 4°C with the remaining cell lysate. Next the membranes were washed extensively five times with 0.1% TBST and then incubated with anti mCherry  or anti-GFP antibody  at RT for 2 h. The membranes were then probed with the appropriate HRP-conjugated secondary antibody  and binding was detected using ECL  in a LAS4000 instrument.g for 15 min at 4°C. Vesicles were then resuspended in the resuspension buffer containing 20 mM HEPES (pH=7.5), 100 mM NaCl and 10 μg of BSA (HiMedia), and incubated with S2R+ cell lysates expressing mCherry::Spo20 or LNS2::GFP for 30 min on ice. Post incubation, liposomes were pelleted as before and the presence of the protein in the pellet or supernatant fraction was analysed by western blotting.Liposomes of the desired composition were made using the phospholipids DOPC  and DOPA. The phospholipids were air-dried, resuspended in buffer containing 20 mM HEPES (pH=7.5), 20 mM KCl and 0.2 M sucrose, sonicated, and freeze-thawed using liquid nitrogen. Post thawing, the solution was mixed with buffer containing 20 mM HEPES (pH=7.5) and 100 mM NaCl, and centrifuged at 200, 000X Anaesthetised flies were immobilized at the end of a pipette tip by applying a drop of colourless nail polish on the proboscis. For recordings, GC 100F-10 borosilicate glass capillaries  were pulled to form electrodes and then filled with 0.8% (w/v) NaCl. The reference electrode was placed on the centre of the eye and the ground electrode on the thorax to obtain voltage changes post stimulation. The protocol for recording involved dark adapting the flies for 5 min initially, following which they were shown green flashes of light for 2 s (ten times), and 12 s of recovery time in dark between the two flashes. Voltage changes obtained were amplified using DAM50 amplifier , and recorded using pCLAMP10.7. Analysis was done using Clampfit 10.7 . For analysis, the average of ten recordings was taken per fly.2 biosensor, tagged to GFP) driven by the transient receptor potential (trp) promoter of flies. Flies were anaesthetised and immobilized at the end of a pipette tip using a drop of colourless nail polish. The flies were then placed on the stage of an Olympus IX71 microscope, and the fluorescent pseudopupil focussed using a 10X objective lens. For imaging the deep pseudopupil, the flies were first adapted to red light for 6 min, following which a blue flash of 90 msec was given. The emitted fluorescence was captured, and its intensity was measured using Image J from NIH . Quantification of the fluorescence intensity was done by measuring the intensity values per unit area of the pseudopupil. The values are represented as mean +/- s.e.m.The imaging is done with flies expressing a single copy of PH-PLCδ::GFP  for P>0.05.Statistical tests were done using unpaired two-tailed


Programmed cell death is regulated by evolutionarily conserved pathways that play critical roles in development and the immune response. A newly recognized pathway for proinflammatory programmed cell death called PANoptosis is controlled by a recently identified cytoplasmic multimeric protein complex named the PANoptosome. The PANoptosome can engage, in parallel, three key modes of programmed cell death—pyroptosis, apoptosis, and necroptosis. The PANoptosome components have been implicated in a wide array of human diseases including autoinflammatory diseases, neurodegenerative diseases, cancer, microbial infections, and metabolic diseases. Here, we review putative components of the PANoptosome and present a phylogenetic analysis of their molecular domains and interaction motifs that support complex assembly. We also discuss genetic data that suggest PANoptosis is coordinated by scaffolding and catalytic functions of the complex components and propose mechanistic models for PANoptosome assembly. Overall, this review presents potential mechanisms governing PANoptosis based on evolutionary analysis of the PANoptosome components. Tak1 in myeloid cells and small molecule inhibitors of TAK1 kinase activity suggested the latter  is an evolutionarily conserved process that plays central roles in maintaining organismal homeostasis. Three key PCD pathways have been studied in great detail—pyroptosis (inflammasome-dependent PCD executed by gasdermin family members), apoptosis (PCD mediated by the apoptosome and executioner caspases), and necroptosis (PCD mediated by RIPK3 and the downstream effector MLKL). Previous studies have provided a foundation for us to understand the active and extensive crosstalk between the inflammasome/pyroptosis and apoptosis and necroptosis  are protected  causes autoinflammation via CASP8- and NLRP3/CASP1-regulated PCD. Combined deletion of Ripk3, Casp1, and Casp8, which blocks all three arms of PANoptosis , rescued the mice from the autoinflammatory condition, while inhibition of a single arm was not sufficient for rescue . The inflammation in Sharpincpdm mice can be rescued by deletion of TNF or expression of kinase-dead RIPK1  released by PANoptotic cells act as alarmins initiating and amplifying the inflammatory response. Overall, the PANoptosome is responsible for driving this inflammatory form of cell death, and defects in PANoptosome components have been implicated in a host of human diseases including autoinflammatory and neurodegenerative diseases, cancer, and susceptibility to pathogenic infections , Table 1Genetic and biochemical data suggest that proteins that are part of the PANoptosome can be generally categorized into three classes—(1) ZBP1 and NLRP3 as putative PAMP and DAMP sensors, (2) ASC and FADD as adaptors, and (3) RIPK1, RIPK3, CASP1, and CASP8 as catalytic effectors. A recent study identified CASP6 as a critical component of the PANoptosome that promotes its assembly by reinforcing the interaction between RIPK3 and ZBP1 in the complex, suggesting a novel scaffold function for this presumed apoptotic executioner caspase  assembly domains, (2) catalytic domains, and (3) sensing domains. Assembly domains include CARD, DEATH, DED, PYD, and RHIM. Catalytic domains are Peptidase_C14 (caspase domain), Pkinase (kinase domain in RIPK3), Pkinase_Tyr (kinase domain in RIPK1), and NACHT (ATPase domain in NLRP3). Sensing domains include z-alpha and LRR_6. We used the Pfam database to look at the evolution of these domains  is in the same branch as the PYD from NLRP3 and ASC. Previous studies have revealed that the PYD of ASC (PYDASC) is required for CASP8 polymerization potentially through a heterotypic interaction between DED2CASP8 and PYDASC  was close to the FADD DED motif (DEDFADD). The homotypic interaction mediated by DEDFADD and DED1CASP8 is known to be important for DISC assembly during extrinsic apoptosis  and one of the RHIM domains of ZBP1 (RHIM2ZBP1) were closer to each other than to CARDASC and CARDCASP1 or RHIM1ZBP1 and RHIM2ZBP1, respectively. This result suggests that upon ligand sensing, ZBP1 may be able to induce assembly of the PANoptosome by recruiting ASC through a heterotypic interaction between RHIM2ZBP1 and CARDASC.PANoptosome components contain a wide range of molecular motifs that can be utilized for assembly of the active complex. However, a clear domain-domain homotypic interaction-based assembly mechanism has not yet been deciphered. Phylogenetic analysis of the assembly domains of the components using Maximum Likelihood Analysis allows a testable model for the mechanism of PANoptosome assembly to be built . Since tZBP1 and CARDASC heterotypic interactions and induce ASC polymerization. PYDASC can then be engaged to recruit NLRP3 into the complex through PYDNLRP3. Since CASP1 activation in IAV-infected cells is strictly dependent on NLRP3, NLRP3 recruitment to the PANoptosome must precede recruitment of CASP1  increases PYDASC polymerization  can recruit RIPK1 and/or RIPK3. CASP8 can be recruited to the complex in two ways—(1) through the DED domain of RIPK1 (DEDRIPK1) interacting with one of the DEDCASP8 domains and/or (2) through PYDASC interacting with DED2CASP8. Future experiments will help define the specific molecular interactions facilitating PANoptosome assembly.These observations suggest a putative assembly mechanism for the ZBP1 PANoptosome involving IAV vRNP sensing by ZBP1 . In thisTwo recent studies have suggested a role for interactions mediated by intrinsically disordered regions (IDR) in inducing PCD (Samir et al., The PANoptosome contains molecules that can induce PCD by multiple mechanisms. This means that a full complex may not need to be assembled before cells undergo cell death. This provides opportunities, and difficulties, for the molecular dissection of PANoptosis. For example, inhibition of one arm of PANoptosis will not be sufficient to prevent cells from dying. This can be exploited to study the ways in which different PCD pathways crosstalk with each other. Additionally, identification of apical sensors that control PANoptosis as a whole will be critical for the modulation of this process. So far ZBP1 is the only apical sensor that has been identified to induce PANoptosis in response to IAV infection (Kuriakose et al., The recent progress in our understanding of the extensive crosstalk between different cell death and signaling cascades unequivocally establishes the existence of multifaceted signaling platforms. The PANoptosome represents one such platform which can engage multiple cell death modalities including pyroptosis, apoptosis, and necroptosis to form the collective inflammatory cell death pathway of PANoptosis. The PANoptosome appears to have evolved to efficiently utilize diverse modes of cellular communication that include domain-based scaffolds and enzyme-based catalytic modifications to engage PANoptosis. Using these diverse cellular mechanisms might be an important way in which the PANoptosome incorporates fundamentally different modes of cell death to orchestrate homeostasis during developmental programs while driving a robust inflammatory form of cell death for immune-mediated protection against microbial infections. Because many of the PANoptosome components have been associated with disease, including autoinflammatory, infectious, and neurodegenerative diseases and cancer, improved understanding of the molecular underpinnings of the PANoptosome will be able to inform the development of targeted inhibitors and activators of inflammatory cell death for therapeutic modulation of inflammation and the immune response.PS performed the phylogenetic and evolutionary analyses. All authors contributed to writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.