Myristoylation Motif Research Articles

Role of molecular mimicry in the SARS-CoV-2-human interactome for pathogenesis of cardiovascular diseases: An update to ImitateDB

Mimicry of host proteins is a strategy employed by pathogens to hijack host functions. Domain and motif mimicry was explored in the experimental and predicted SARS-CoV-2-human interactome. The host first interactor proteins were also added to capture the continuum of the interactions. The domains and motifs of the proteins were annotated using NCBI CD Search and ScanProsite, respectively. Host and pathogen proteins with a common host interactor and similar domain/motif constitute a mimicry pair indicating global structural similarity (domain mimicry pair; DMP) or local sequence similarity (motif mimicry pair; MMP). 593 DMPs and 7,02,472 MMPs were determined. AAA, DEXDc and Macro domains were frequent among DMPs whereas glycosylation, myristoylation and RGD motifs were abundant among MMP. The proteins involved in mimicry were visualised as a SARS-CoV-2 mimicry interaction network. The host proteins were enriched in multiple CVD pathways indicating the role of mimicry in COVID-19 associated CVDs. Bridging nodes were identified as potential drug targets. Approved antihypertensive and anti-inflammatory drugs are proposed for repurposing against COVID-19 associated CVDs. The SARS-CoV-2 mimicry data has been updated in ImitateDB (http://imitatedb.sblab-nsit.net/SARSCoV2Mimicry). Determination of key mechanisms, proteins, pathways, drug targets and repurposing candidates is critical for developing therapeutics for SARS CoV-2 associated CVDs.

Structural and Mechanistic Studies of the Rare Myristoylation Signal of the Feline Immunodeficiency Virus

All retroviruses encode a Gag polyprotein containing an N-terminal matrix domain (MA) that anchors Gag to the plasma membrane and recruits envelope glycoproteins to virus assembly sites. Membrane binding by the Gag protein of HIV-1 and most other lentiviruses is dependent on N-terminal myristoylation of MA by host N-myristoyltransferase enzymes (NMTs), which recognize a six-residue “myristoylation signal” with consensus sequence: M1GXXX[ST]. For unknown reasons, the feline immunodeficiency virus (FIV), which infects both domestic and wild cats, encodes a non-consensus myristoylation sequence not utilized by its host or by other mammals (most commonly: M1GNGQG). To explore the evolutionary basis for this sequence, we compared the structure, dynamics, and myristoylation properties of native FIV MA with a mutant protein containing a consensus feline myristoylation motif (MANOS) and examined the impact of MA mutations on virus assembly and ability to support spreading infection. Unexpectedly, myristoylation efficiency of MANOS in Escherichia coli by co-expressed mammalian NMT was reduced by ~70% compared to the wild-type protein. NMR studies revealed that residues of the N-terminal myristoylation signal are fully exposed and mobile in the native protein but partially sequestered in the MANOS chimera, suggesting that the unusual FIV sequence is conserved to promote exposure and efficient myristoylation of the MA N terminus. In contrast, virus assembly studies indicate that the MANOS mutation does not affect virus assembly, but does prevent virus spread, in feline kidney cells. Our findings indicate that residues of the FIV myristoylation sequence play roles in replication beyond NMT recognition and Gag–membrane binding.

Identification of Protein Motifs in Phytoplasma Associated with Root (Wilt) Disease of Coconut (Cocos nucifera) Using an Improved Statistical Measure

Protein motif is a conserved short region within larger sequence, and it provides an important way to get functional or structural information about a noble protein sequence. This study primarily provides a new approach to identify motifs which are considered to play an important role to determine functional or structural class of any protein. Coconut palm (Cocos nucifera L.) root (wilt) disease (CRWD) became a serious concern for coconut cultivation in the coastal region of South India. It has already been reported that species of the phytoplasma (-belonging to ribosomal group 16SrXI) is primarily associated with CRWD. In this study, our objective is to modify an existing motif identification algorithm, and also identify motifs that describe their conserved region within a CRWD phytoplasma protein sequence. Accordingly, the existing information content measure formulas for a no-gapped and gapped aligned sequence set were re-formulated. Further, an improvement on both these existing measures has been vied by incorporating prior information of BLOSUM90 substitution matrix. Results showed that proposed modifications could successfully identify N-terminal myristoylation motif within an alignment matrix of Arabidopsis thaliana sequences; concurrently our modification has also been validated. Both the approaches, namely frequency- and BLOSUM90-based information content, have been compared using the observed results, in terms of identified motifs. Finally, we could also test this noble BLOSUM90-based approach to correctly predict conserved regions at locations, (163–171) and (117–138), in an aligned secA gene sequence set of phytoplasma, an etiological agent of CRWD.

Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses

Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.

Development of a bicistronic expression system in the branchiopod crustacean Daphnia magna.

The viral 2A peptides have recently been used for bicistronic expression in various organisms. In this system, a single mRNA that codes for two proteins flanking the 2A peptide can be translated simultaneously into each protein by ribosomal skipping at this peptide sequence. Here, we tested the function of the Thosea asigna insect virus 2A (T2A) peptide in the branchiopod crustacean Daphnia magna-an emerging model of evolutionary developmental biology. First, we used transgenic Daphnia that expresses a potential bicistronic RNA containing mCherry and histone H2B- green fluorescent protein (GFP) open reading frames upstream and downstream of the T2A sequence, respectively. Microscopic observation revealed difference of localization of the two proteins in the cell, homogenous distribution of mCherry and nuclear localization of H2B-GFP. Second, we changed localization of mCherry from cytoplasm to plasma membrane by attachment of a consensus myristoylation motif in the bicistronic reporter. RNA that codes for this new bicistronic reporter was injected into eggs. At gastrulation stage, we found spectrally distinct fluorescence with enough intensity and resolution to detect membrane localized mCherry and nuclear GFP. These results indicate that the T2A peptide functions in D. magna and T2A-mediated bicistronic expression would be a promising tool for evo-devo studies of this species.

Delivery of gene targeting siRNAs to breast cancer cells using a multifunctional peptide complex that promotes both targeted delivery and endosomal release.

RNA interference has been used to dissect the importance of individual gene products in various human disease processes, including cancer. Small-interfering RNA, or siRNA, is one of the tools utilized in this regard, but specially-designed delivery agents are required to allow the siRNA to gain optimal access to the cell interior. Our laboratory has utilized two different siRNA-binding delivery peptides containing a polyarginine core, and modified by myristoylation and targeting motifs (iRGD or Lyp-1). A third peptide was designed to assist with endosomal release. Various ratios of the peptides and siRNA were combined and assayed for the ability to form stable complexes, and optimized ratios were determined. The complexes were found to form particles, with the majority having a diameter of 100–300 nm, as visualized by electron microscopy. These siRNA complexes have enhanced protection from nucleases present in serum, as compared to “naked” unprotected siRNA. The particles were internalized by the cells and could be detected in the cell cytoplasm by confocal fluorescence microscopy. In functional assays, peptide/siRNA complexes were shown to cause the knock down of corresponding targeted proteins. The peptide with the LyP-1 targeting motif was more effective at knockdown in MDA-MB-231 breast cancer cells than the peptide with the iRGD motif. Inclusion of the endosomal release peptide in the complexes greatly enhanced the peptide/siRNA effects. Peptide/siRNA complexes simultaneously targeting Stat3 and c-Myc caused a marked reduction in anchorage-independent growth, a property correlated with tumorigenicity. This study demonstrates the ability of a peptide-based siRNA-delivery system to deliver siRNA into breast cancer cells and cause both protein knockdown and suppression of the malignant phenotype. Such peptide complexes are likely to become highly useful siRNA-delivery vehicles for the characterization, and potentially for the treatment, of human cancer.

Glutathione peroxidase-like enzymes cover five distinct cell compartments and membrane surfaces in Arabidopsis thaliana.

Glutathione peroxidase-like enzymes (GPXLs) constitute a family of eight peroxidases in Arabidopsis thaliana. In contrast to the eponymous selenocysteine glutathione peroxidases in mammalian cells that use glutathione as electron donor, GPXLs rely on cysteine instead of selenocysteine for activity and depend on the thioredoxin system for reduction. Although plant GPXLs have been implicated in important agronomic traits such as drought tolerance, photooxidative tolerance and immune responses, there remain major ambiguities regarding their subcellular localization. Because their site of action is a prerequisite for an understanding of their function, we investigated the localization of all eight GPXLs in stable Arabidopsis lines expressing N-terminal and C-terminal fusions with redox-sensitive green fluorescent protein 2 (roGFP2) using confocal microscopy. GPXL1 and GPXL7 were found in plastids, while GPXL2 and GPXL8 are cytosolic nuclear. The N-terminal target peptide of GPXL6 is sufficient to direct roGFP2 into mitochondria. Interestingly, GPXL3, GPXL4 and GPXL5 all appear to be membrane bound. GPXL3 was found exclusively in the secretory pathway where it is anchored by a single N-terminal transmembrane domain. GPXL4 and GPXL5 are anchored to the plasma membrane. Presence of an N-terminal myristoylation motif and genetic disruption of membrane association through targeted mutagenesis point to myristoylation as essential for membrane localization.

Characterization of the calcineurin B-Like (CBL) gene family in maize and functional analysis of ZmCBL9 under abscisic acid and abiotic stress treatments

In plants, calcineurin B-like proteins (CBLs) play crucial roles in regulating calcium-signaling in response to various abiotic stresses by interacting with specific CBL-interacting protein kinases (CIPKs). However, the identities and functions of CBL gene family members in maize are largely unknown. Here, we identified from the maize genome 12 CBL genes. All 12 CBLs have conserved EF-hand domains, and half harbor myristoylation motifs. We further characterized the function of one CBL gene, ZmCBL9, which can be induced by salt, dehydration, glucose and abscisic acid (ABA) treatments. Overexpression of ZmCBL9 enhanced resistance or tolerance to ABA, glucose, salt and osmotic stress in Arabidopsis and complemented the hypersensitive phenotype of the Arabidopsis cbl9 mutant in response to ABA and abiotic stress. The ZmCBL9 gene negatively regulates the expression of genes in the ABA signaling, biosynthesis and catabolism pathways. Moreover, the ZmCBL9 protein is found to interact with eight maize CIPKs and these ZmCIPK genes were up-regulated by different stress treatments, including salt, dehydration, glucose, low potassium and ABA. These results suggest that ZmCBL9 may interact with various ZmCIPKs to regulate the abiotic stress and ABA response signaling in plants.

Translation, modification and cellular distribution of two AC4 variants of African cassava mosaic virus in yeast and their pathogenic potential in plants

Plant infecting geminiviruses encode a small (A)C4 protein within the open reading frame of the replication-initiator protein. In African cassava mosaic virus, two in-frame start codons may be used for the translation of a longer and a shorter AC4 variant. Both were fused to green fluorescent protein or glutathione-S-transferase genes and expressed in fission yeast. The longer variant accumulated in discrete spots in the cytoplasm, whereas the shorter variant localized to the plasma membrane. A similar expression pattern was found in plants. A myristoylation motif may promote a targeting of the shorter variant to the plasma membrane. Mass spectrometry analysis of the yeast-expressed shorter variant detected the corresponding myristoylation. The biological relevance of the second start codon was confirmed using mutated infectious clones. Whereas mutating the first start codon had no effect on the infectivity in Nicotiana benthamiana plants, the second start codon proved to be essential.

Plasmodium Rab5b is secreted to the cytoplasmic face of the tubovesicular network in infected red blood cells together with N-acylated adenylate kinase 2.

BackgroundRab5 GTPase regulates membrane trafficking between the plasma membrane and endosomes and harbours a conserved C-terminal isoprenyl modification that is necessary for membrane recruitment. Plasmodium falciparum encodes three Rab5 isotypes, and one of these, Rab5b (PfRab5b), lacks the C-terminal modification but possesses the N-terminal myristoylation motif. PfRab5b was reported to localize to the parasite periphery. However, the trafficking pathway regulated by PfRab5b is unknown.MethodsA complementation analysis of Rab5 isotypes was performed in Plasmodium berghei. A constitutively active PfRab5b mutant was expressed under the regulation of a ligand-dependent destabilization domain (DD)-tag system in P. falciparum. The localization of PfRab5b was evaluated after removing the ligand followed by selective permeabilization of the membrane with different detergents. Furthermore, P. falciparum N-terminally myristoylated adenylate kinase 2 (PfAK2) was co-expressed with PfRab5b, and trafficking of PfAK2 to the parasitophorous vacuole membrane was examined by confocal microscopy.ResultsPfRab5b complemented the function of PbRab5b, however, the conventional C-terminally isoprenylated Rab5, PbRab5a or PbRab5c, did not. The constitutively active PfRab5b mutant localized to the cytosol of the parasite and the tubovesicular network (TVN), a region that extends from the parasitophorous vacuole membrane (PVM) in infected red blood cells (iRBCs). By removing the DD-ligand, parasite cytosolic PfRab5b signal disappeared and a punctate structure adjacent to the endoplasmic reticulum (ER) and parasite periphery accumulated. The peripheral PfRab5b was sensitive to extracellular proteolysis after treatment with streptolysin O, which selectively permeabilizes the red blood cell plasma membrane, indicating that PfRab5b localized on the iRBC cytoplasmic face of the TVN. Transport of PfAK2 to the PVM was abrogated by overexpression of PfRab5b, and PfAK2 accumulated in the punctate structure together with PfRab5b.ConclusionN-myristoylated Plasmodium Rab5b plays a role that is distinct from that of conventional mammalian Rab5 isotypes. PfRab5b localizes to a compartment close to the ER, translocated to the lumen of the organelle, and co-localizes with PfAK2. PfRab5b and PfAK2 are then transported to the TVN, and PfRab5b localizes on the iRBC cytoplasmic face of TVN. These data demonstrate that PfRab5b is transported from the parasite cytosol to TVN together with N-myristoylated PfAK2 via an uncharacterized membrane-trafficking pathway.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-016-1377-4) contains supplementary material, which is available to authorized users.

Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance.

Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato (Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca(2+) binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance.

Direct visualization of cGMP microdomains in adult cardiomyocytes

Results Cardiomyocytes were isolated from the transgenic mice expressing the cytosolic red cGES-DE5 sensor [1]. Using the previously established scanning ion conductance microscopy (SICM)/FRET method [2], local application of Atrial Natriuretic Peptide (ANP) at T-tubules and crests revealed the exclusive localization of guanylyl cyclase A (GC-A) in T-tubules. In contrast, guanylyl cyclase B (GC-B), the receptor for the C-type Natriuretic Peptide (CNP), was more evenly distributed across the membrane. Further, two differentially targeted cGMP sensors were generated. One of them was targeted to the caveolin-rich membrane microdomains via a palmitoylation and myristoylation motif (pmDE5) and another one to the sarcoplasmic reticulum by the fusion with phospholamban (DE5-PLN). FRET measurements in cardiomyocytes from these transgenic mice revealed close association of the GC-A with the caveolin-rich membrane microdomains, as well as a CNP, but not ANP-dependent regulation of cGMP levels at the sarcoplasmic reticulum.

Cloning and sequence analysis of Interleukin (IL)-2, IL-4, IL-10, and IL-18 of Marwari horse

Cytokines play very important roles in the development of protective immune responses against a variety of pathogens. During the last decade rapid progress has been made in the cloning and characterization of cytokines from livestock and companion animals. However, no such work was carried out on cytokines of Indian horse breed. The present work was undertaken with the objective of generating sequence information of cytokines of Marwari horse. The cDNAs encoding interleukin (IL)-2, IL-4, IL-10 and IL-18 of Marwari horse were cloned, sequenced and subsequently compared with respective interleukin sequences of mammalian species including zebra, donkey, cattle, buffalo, sheep, goat, camel and pig available in GenBank database. Horse IL-10 and IL-18 shows nearly 90% sequence homology both at nucleotide and amino acid level with major livestock species. But horse IL-2 and IL-4 had significant sequence divergence with corresponding interleukins of domestic animals. Structural and functional conservation of horse interleukins in respect of cysteine residues, glycosylation, myristoylation, and phosphorylation motifs revealed that IL-10 and IL-18 molecules are evolutionary conserved across species. Nucleotide and amino acid sequences identity and phylogenetic analyses of the 4 cytokine genes in the present study indicated that horse cytokines are closely related to that of pig and camel cytokines.

Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis.

The cilium both releases and binds to extracellular vesicles (EVs). EVs may be used by cells as a form of intercellular communication and mediate a broad range of physiological and pathological processes. The mammalian polycystins (PCs) localize to cilia, as well as to urinary EVs released from renal epithelial cells. PC ciliary trafficking defects may be an underlying cause of autosomal dominant polycystic kidney disease (PKD), and ciliary-EV interactions have been proposed to play a central role in the biology of PKD. In Caenorhabditis elegans and mammals, PC1 and PC2 act in the same genetic pathway, act in a sensory capacity, localize to cilia, and are contained in secreted EVs, suggesting ancient conservation. However, the relationship between cilia and EVs and the mechanisms generating PC-containing EVs remain an enigma. In a forward genetic screen for regulators of C. elegans PKD-2 ciliary localization, we identified CIL-7, a myristoylated protein that regulates EV biogenesis. Loss of CIL-7 results in male mating behavioral defects, excessive accumulation of EVs in the lumen of the cephalic sensory organ, and failure to release PKD-2::GFP-containing EVs to the environment. Fatty acylation, such as myristoylation and palmitoylation, targets proteins to cilia and flagella. The CIL-7 myristoylation motif is essential for CIL-7 function and for targeting CIL-7 to EVs. C. elegans is a powerful model with which to study ciliary EV biogenesis in vivo and identify cis-targeting motifs such as myristoylation that are necessary for EV-cargo association and function.

A Truncated Fragment of Src Protein Kinase Generated by Calpain‐mediated Cleavage is a Mediator of Neuronal Death in Excitotoxicity.

Excitotoxicity resulting from overstimulation of glutamate receptors is a major cause of neuronal death in cerebral ischemic stroke. The overstimulated ionotropic glutamate receptors exert their neurotoxic effects in part by overactivation of calpains, which induce neuronal death by catalyzing limited proteolysis of specific cellular proteins. Here, we report that in cultured cortical neurons and in vivo in a rat model of focal ischemic stroke, the tyrosine kinase Src is cleaved by calpains at a site in the N‐terminal unique domain. This generates a truncated Src fragment of ~52 kDa, which we localized predominantly to the cytosol. A cell membrane‐permeable fusion peptide derived from the unique domain of Src prevents calpain from cleaving Src in neurons and protects against excitotoxic neuronal death. To explore the role of the truncated Src fragment in neuronal death, we expressed a recombinant truncated Src fragment in cultured neurons and examined how it affects neuronal survival. Expression of this fragment, which lacks the myristoylation motif and unique domain, was sufficient to induce neuronal death. Furthermore, inactivation of the prosurvival kinase Akt is a key step in its neurotoxic signaling pathway. Because Src maintains neuronal survival, our results implicate calpain cleavage as a molecular switch converting Src from a promoter of cell survival to a mediator of neuronal death in excitotoxicity. Besides unveiling a new pathological action of Src, our discovery of the neurotoxic action of the truncated Src fragment suggests new therapeutic strategies with the potential to minimize brain damage in ischemic stroke

Liaison between myristoylation and cryptic EF-hand motif confers Ca(2+) sensitivity to neuronal calcium sensor-1.

Many members of the neuronal calcium sensor (NCS) protein family have a striking coexistence of two characteristics, that is, N-myristoylation and the cryptic EF-1 motif. We investigated the rationale behind this correlation in neuronal calcium sensor-1 (NCS-1) by restoring Ca(2+) binding ability of the disabled EF-1 loop by appropriate mutations. The concurrence of canonical EF-1 and N-myristoylation considerably decreased the overall Ca(2+) affinity, conformational flexibility, and functional activation of downstream effecter molecules (i.e., PI4Kβ). Of a particular note, Ca(2+) induced conformational change (which is the first premise for a CaBP to be considered as sensor) is considerably reduced in myristoylated proteins in which Ca(2+)-binding to EF-1 is restored. Moreover, Ca(2+), which otherwise augments the enzymatic activity of PI4Kβ (modulated by NCS-1), leads to a further decline in the modulated PI4Kβ activity by myristoylated mutants (with canonical EF-1) pointing toward a loss of Ca(2+) signaling and specificity at the structural as well as functional levels. This study establishes the presence of the strong liaison between myristoylation and cryptic EF-1 in NCS-1. Breaking this liaison results in the failure of Ca(2+) specific signal transduction to downstream effecter molecules despite Ca(2+) binding. Thus, the EF-1 disability is a prerequisite in order to append myristoylation signaling while preserving structural robustness and Ca(2+) sensitivity/specificity in NCS-1.

Analysis of Presenilin 1 and 2 interacting proteins in mouse cerebral cortex during development

In our previous report, we showed that Presenilin (PS)1 and 2 have differential expression profile from early embryonic stages till adulthood in mouse cerebral cortex, suggesting that both of these proteins are crucial for brain development. Genetic manipulation studies have also shown the involvement of PS1 in brain development, but PS2 remains largely unexplored. In order to understand how PS1 and 2 mediate developmental functions, we have investigated the interaction of PS1 and 2 with proteins of mouse cerebral cortex during development. Co-immunoprecipitation (Co-IP) combined with MALDI-MS/MS analysis revealed 12 interacting partners of PS1 and 11 partners of PS2. The interacting proteins were different for PS1 and 2, and involved in cell division, glycolysis, cell adhesion and protein trafficking. Densitometric analysis of protein bands visualized after SDS-PAGE separation of Co-IP proteins revealed variation in their amount and degree of interaction during different developmental stages of mice. Further, immunoblot based validation of PS1 interacting protein Notch-1 showed maximum interaction at embryonic day (E) 12.5, decline at E18.5, upregulation from postnatal day 0 (P0) to P20 and thereafter reduction at P45 and 20 weeks. In-silico analysis of PS and its interacting proteins indicated conformation based interaction through common type of secondary structures having alpha helical, extended beta strand and random coil, and CK2, PKC phosphorylation and myristoylation motifs. Taken together, our study showed that PS1 and PS2 interact to varying extent with different proteins of mouse cerebral cortex and suggests their interaction based on specific conformation and involvement in diverse functions essential for the brain development.

Plasmodium falciparum Rab5B is an N-terminally myristoylated Rab GTPase that is targeted to the parasite's plasma and food vacuole membranes.

Plasmodium falciparum (Pf) has a family of 11 Rab GTPases to regulate its vesicular transport. However, PfRab5B is unique in lacking a C-terminal geranyl-geranylation motif, while having N-terminal palmitoylation and myristoylation motifs. We show that the N-terminal glycine is required for PfRab5B myristoylation in vitro and when an N-terminal PfRab5B fragment possessing both acylation motifs is fused to GFP and expressed in transgenic P. falciparum parasites, the chimeric PfRab5B protein localizes to the plasma membrane. Upon substitution of the modified glycine by alanine the staining becomes diffuse and GFP is found in soluble subcellular fractions. Immuno-electron microscopy shows endogenous PfRab5B decorating the parasite's plasma and food vacuole membranes. Using reverse genetics rab5b couldn't be deleted from the haploid genome of asexual blood stage P. berghei parasites. The failure of PbRab5A or PbRab5C to complement for loss of PbRab5B function indicates non-overlapping roles for the three Plasmodium Rab5s, with PfRab5B involved in trafficking MSP1 to the food vacuole membrane and CK1 to the plasma membrane. We discuss similarities between Plasmodium Rab5B and Arabidopsis thaliana ARA6, a similarly unusual Rab5-like GTPase of plants.

The Xanthomonas campestris Type III Effector XopJ Targets the Host Cell Proteasome to Suppress Salicylic-Acid Mediated Plant Defence

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.