A-kinase anchoring proteins are enriched in the central pair microtubules of motile cilia in Chlamydomonas reinhardtii.

Cilia are microtubule-based sensory organelles present in a number of eukaryotic cells. Mutations in the genes encoding ciliary proteins cause ciliopathies in humans. A-kinase anchoring proteins (AKAPs) tether ciliary signaling proteins such as protein kinase A (PKA). The dimerization and docking domain (D/D) on the RIIα subunit of PKA interacts with AKAPs. Here, we show that AKAP240 from the central-pair microtubules of Chlamydomonas reinhardtii cilia uses two C-terminal amphipathic helices to bind to its partner FAP174, an RIIα-like protein with a D/D domain at the N-terminus. Co-immunoprecipitation using anti-FAP174 antibody with an enriched central-pair microtubule fraction isolated seven interactors whose mass spectrometry analysis revealed proteins from the C2a (FAP65, FAP70, and FAP147) and C1b (CPC1, HSP70A, and FAP42) microtubule projections and FAP75, a protein whose sub-ciliary localization is unknown. Using RII D/D and FAP174 as baits, we identified two additional AKAPs (CPC1 and FAP297) in the central-pair microtubules.

Isolation and characterization of extracellular vesicles from Fusarium oxysporum f. sp. cubense, a banana wilt pathogen.

Fusarium wilt of banana is a destructive widespread disease caused by Fusarium oxysporum f. sp. cubense (Foc) that ravaged banana plantations globally, incurring huge economic losses. Current knowledge demonstrates the involvement of several transcription factors, effector proteins, and small RNAs in the Foc-banana interaction. However, the precise mode of communication at the interface remains elusive. Cutting-edge research has emphasized the significance of extracellular vesicles (EVs) in trafficking the virulent factors modulating the host physiology and defence system. EVs are ubiquitous inter- and intra-cellular communicators across kingdoms. This study focuses on the isolation and characterization of Foc EVs from methods that make use of sodium acetate, polyethylene glycol, ethyl acetate, and high-speed centrifugation. Isolated EVs were microscopically visualized using Nile red staining. Further, the EVs were characterized using transmission electron microscopy, which revealed the presence of spherical, double-membrane, vesicular structures ranging in size from 50 to 200 nm (diameter). The size was also determined using the principle based on Dynamic Light Scattering. The Foc EVs contained proteins that were separated using SDS-PAGE and ranged between 10 and 315 kDa. Mass spectrometry analysis revealed the presence of EV-specific marker proteins, toxic peptides, and effectors. The Foc EVs were found to be cytotoxic, whose toxicity increased with EVs isolated from the co-culture preparation. Taken together, a better understanding of Foc EVs and their cargo will aid in deciphering the molecular crosstalk between banana and Foc.

Appearing and disappearing acts of cilia.

The past few decades have seen a rise in research on vertebrate cilia and ciliopathy, with interesting collaborations between basic and clinical scientists. This work includes studies on ciliary architecture, composition, evolution, and organelle generation and its biological role. The human body has cells that harbour any of the following four types of cilia: 9+0 motile, 9+0 immotile, 9+2 motile, and 9+2 immotile. Depending on the type, cilia play an important role in cell/fluid movement, mating, sensory perception, and development. Defects in cilia are associated with a wide range of human diseases afflicting the brain, heart, kidneys, respiratory tract, and reproductive system. These are commonly known as ciliopathies and affect millions of people worldwide. Due to their complex genetic etiology, diagnosis and therapy have remained elusive. Although model organisms like Chlamydomonas reinhardtii have been a useful source for ciliary research, reports of a fascinating and rewarding translation of this research into mammalian systems, especially humans, are seen. The current review peeks into one of the complex features of this organelle, namely its birth, the common denominators across the formation of both 9+0 and 9+2 ciliary types, the molecules involved in ciliogenesis, and the steps that go towards regulating their assembly and disassembly.

Effect of UV Stress on the Structure and Function of Pro-apoptotic Bid and Anti-apoptotic Bcl-xl proteins.

Ultraviolet C (UV-C) radiation induces apoptosis in mammalian cells via the mitochondrion-mediated pathway. The Bcl-2 family of proteins are the regulators of the mitochondrial pathway of apoptosis and appears responsive to UV-C radiation. It is unknown how the structure and, effectively, the function of these proteins are directly impacted by UV-C exposure. Here, we present the effect of UV-C irradiation on the structure and function of pro-apoptotic Bid-FL and anti-apoptotic Bcl-xlΔC proteins. Using a variety of biophysical tools, we show that, following UV-C irradiation, the structures of Bcl-xlΔC and Bid-FL are irreversibly altered. Bcl-xLΔC is found to be more sensitive to UV stress than Bid-FL Interestingly, UV-C exposure shows dramatic chemical shift perturbations in consequence of dramatic structural perturbations (α-helix to ß-sheet) in the BH3- binding region, a crucial segment of Bcl-xlΔC. Furter it has been shown that UV-exposed Bcl-xlΔC has reduced efficacy of its interactions with pro-apoptotic tBid.

Strategic optimization of conditions for the solubilization of GST-tagged amphipathic helix-containing ciliary proteins overexpressed as inclusion bodies in E. coli.

Expression of affinity-tagged recombinant proteins for crystallography, protein-protein interaction, antibody generation, therapeutic applications, etc. mandates the generation of high-yield soluble proteins. Although recent developments suggest the use of yeast, insect, and mammalian cell lines as protein expression platforms, Escherichia coli is still the most popular, due mainly to its ease of growth, feasibility in genetic manipulation and economy. However, some proteins have a spontaneous tendency to form inclusion bodies (IBs) when over-expressed in bacterial expression systems such as E. coli, thus posing a challenge in purification and yield. At times, small peptides undergo degradation during protein production and hence using suitable tags could circumvent the problem. Although several independent techniques have been used to solubilize IBs, these cannot always be applied in a generic sense. Although tagging a GST moiety is known to enhance the solubility of fusion proteins in E. coli, resulting in yields of 10-50 mg/L of the culture, the inherent nature of the protein sequence at times could lead to the formation of IBs. We have been working on a Myc Binding Protein-1 orthologue, viz. Flagellar Associated Protein 174 (FAP174) from the axoneme of Chlamydomonas reinhardtii that binds to an A-Kinase Anchoring Protein 240 (AKAP240) which has been annotated as Flagellar Associated Protein 65 (FAP65). Using an in-silico approach, we have identified two amphipathic helices on FAP65 (CrFAP65AH1 and CrFAP65AH2) that are predicted to bind to FAP174. To test this prediction, we have cloned the GST-tagged peptides, and overexpressed them in E. coli that have resulted in insoluble IBs. The yields of these over-expressed recombinant proteins dropped considerably due to IB formation, indicating aggregation. An integrated approach has been used to solubilize four highly hydrophobic polypeptides, viz. two amphipathic helices and the respective proline variants of FAP65. For solubilizing these polypeptides, variables such as non-denaturing detergents (IGEPAL CA-630), changing the ionic strength of the cell lysis and solubilization buffer, addition of BugBuster®, diluting the cell lysate and sonication were introduced. Our statistically viable results yielded highly soluble and functional polypeptides, indiscreet secondary structures, and a yield of ~ 20 mg/L of the E. coli culture. Our combinatorial strategy using chemical and physical methods to solubilize IBs could prove useful for hydrophobic peptides and proteins with amphipathic helices.

In silico insight of cell-death-related proteins in photosynthetic cyanobacteria.

Cyanobacteria are a large group of ubiquitously found photosynthetic prokaryotes that are constantly exposed to different kinds of stressors of varying intensities and seem to overcome these in a precise and regulated manner. However, a high dose and duration of given stress induce cell death in a few select cyanobacteria, mainly to protect other cells (altruism). Despite the recent findings for the presence of biochemical and molecular hallmarks of cell death in cyanobacteria, it is yet a sketchily understood phenomenon. Regulation of metacaspase-like genes during Programmed Cell Death suggests it to be a genetically controlled mechanism like other eukaryotes. In addition to providing a comprehensive understanding of the current status of cell death in cyanobacteria, this review has used in silico analyses to directly compare the existence of some important molecular players operating in the intrinsic and extrinsic apoptotic pathways. Phylogenetic trees for all sequences indicate a cluster with a common ancestry and also a divergence from sequences of eukaryotic origin. To the best of our knowledge, such a comparison (except for orthocaspases) has not been attempted earlier and hopes to encourage workers in the field to investigate this altruistic phenomenon in detail.

Curcumin, a potential initiator of apoptosis via direct interactions with Bcl-xL and Bid.

Apoptosis is a naturally occurring process during the growth and development of multicellular organisms and is increasingly active during times of cellular stress such as in response to intracellular DNA damage when removal of the host cell is paramount to prevent cancer. Unfortunately, once formed, cancer cells become impervious to apoptosis, creating a desperate need to identify an approach to induce apoptosis in these cells. An attractive option is to focus efforts on developing and locating compounds which activate apoptosis using natural compounds. Curcumin is a natural component in turmeric and is well-known for its pharmacological effects in preventing and combating many ailments and has been shown to decrease the rapid proliferation of a wide variety of tumor cells. However, to date, the apoptotic intermediates and interactions through which curcumin exerts its cytotoxic effects are unknown. Motivated by reports linking the intracellular modulation of the concentrations of Bid and Bcl-xL, following curcumin administration to cancer cells, we set out to probe for potential intermolecular interactions of these proteins with curcumin. Using several biophysical techniques, most notably, fluorescence, circular dichroism and nuclear magnetic resonance spectroscopy, we reveal binding interactions of curcumin with both Bcl-xLΔC and full-length Bid (Bid-FL) and prove that this binding is hydrophobically driven and localized to well-known functional regions of each protein. Specifically, our NMR studies show that while Bid-FL interacts with curcumin through its hydrophobic and pore forming helices (α6-α7), Bcl-xLΔC interacts with curcumin via its BH3 binding pocket (α2-α3-α4-α5), a critical region for mediating apoptosis.

A potential screening method for epigenetic drugs: uncovering stress-induced gene silencing in Chlamydomonas.

Histone modifications and DNA methylation together govern promoter availability, thereby influencing gene expression. This study queries the unicellular chlorophyte, Chlamydomonas reinhardtii using a three step "epigenetic assay" design to phenotypically track the variegation of a randomly integrated Paromomycin resistance transgene(s) (PmR). Based on its position of integration, the PmR gene expression hinged on two epigenetic hallmarks: the spreading of heterochromatin, and the transmissible memory of epigenetic states across generations. Using a spot-dilution analysis, the loss of antibiotic resistance phenotype was scored from 0 to 4, four being maximally silenced. Appropriate construct designs were used to demonstrate that the cis-spread of heterochromatin could be interfered with a stronger euchromatic barrier (TUB2 promoter). When assayed for metal ion stress, a combination of Mn deficiency with excess Cu or Zn stress was shown to induce gene silencing in Chlamydomonas. Cu stress resulted in the accumulation of intracellular ROS, while Zn stress elevated the sensitivity to ROS. As proof of functional conservation, mammalian epigenetic drugs demonstrably interfered with stress-induced gene silencing. Finally, a selected group of transgenic clones responsive to HDACi sodium butyrate, when tested in a gradient plate format showed similarity in phenotype to the plant-derived compound cinnamic acid. This indicated a possible commonality in their mode of action, unlike curcumin which might have a different mechanism. Thus, using binned libraries, based on a common set of responses to known drugs, a cost-effective high-throughput screening strategy for epigenetically active compounds from plants or other sources is described.

Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense.

Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5' ends in close proximity. These genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. This intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely ß-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. Characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.

Vertical flow paper-based plasmonic device for cysteine detection.

Cystinuria, is an autosomal recessive genetic disorder involving increasingly high levels of poorly soluble cysteine in urine leading to formation of stones. Developing a facile, low-cost, point-of-care and selective sensor for diagnosis of cysteine is imperative. Accordingly, for the detection of cysteine, the present study demonstrates an inexpensive colorimetric, paper-based vertical flow plasmonic micro-well device with a two-minute turn-around time. The method encompasses the use of microbially-synthesized silver nanoparticles (AgNPs) that change from light brown / yellow to dark brown upon binding with Sulphur present in cysteine. This technique allows for visual detection up to 1 × 10-5 mM cysteine and can be easily offered as a rapid diagnostic test even at setups with minimal resources.

A micro-Raman and chemometric study of urinary tract infection-causing bacterial pathogens in mixed cultures.

Detection of urinary tract infection (UTI)-causing bacteria uses conventional time-consuming microbiological techniques. The current need is to use a fast and reliable method of bacterial identification. In order to unambiguously distinguish the UTI-causing five bacterial species used in the current study, micro-Raman spectra were obtained from a home-assembled micro-Raman system and analyzed by multivariate statistical techniques such as principal component analysis (PCA), partial least square-discriminate analysis (PLS-DA), and support vector machine (SVM). Also, the micro-Raman spectra recorded from samples containing two and three bacterial species were tested and validated against the aforementioned calibration models using PLS-DA and SVM. The prediction accuracies of up to 73 and 89% were achieved with PLS-DA and SVM, respectively. Taken together, the present study depicts the capturing of unique micro-Raman spectral features manifesting from the biochemical content of each bacterium. Also, micro-Raman spectroscopy combined with multivariate data analysis can therefore be a reliable and faster technique for the diagnosis of UTI-causing bacteria. Graphical Abstract.

Defects in the ratio of the dynein isoform, DHC11 in the long-flagella mutants of Chlamydomonas reinhardtii.

The long-flagella mutants (lf1, lf2, lf3 and lf4) of Chlamydomonas reinhardtii are defective in proteins that are required for the assembly of normal flagella, their phenotype being long flagella. In a previous study, we biophysically characterized these mutants for their waveform patterns, swimming speeds, beat frequencies and correlated these parameters with their flagellar lengths. We found an anomaly in this correlation and set out to explore the underlying molecular significance, if any. The diverse inner dynein isoforms are the flagellar motors that convert the chemical energy of ATP into the mechanical energy of motility; we probed the presence of one of these isoforms (DHC11, which might help in bend initiation) in the lf mutants and compared it with the wild-type. Our studies show that the ratio of DHC11 is defective in the long-flagella mutants of Chlamydomonas reinhardtii.

Myc-binding protein orthologue interacts with AKAP240 in the central pair apparatus of the Chlamydomonas flagella.

BACKGROUND: Flagella and cilia are fine thread-like organelles protruding from cells that harbour them. The typical '9 + 2' cilia confer motility on these cells. Although the mechanistic details of motility remain elusive, the dynein-driven motility is regulated by various kinases and phosphatases. A-kinase anchoring proteins (AKAPs) are scaffolds that bind to a variety of such proteins. Usually, they are known to possess a dedicated domain that in vitro interacts with the regulatory subunits (RI and RII) present in the cAMP-dependent protein kinase (PKA) holoenzyme. These subunits conventionally harbour contiguous stretches of a.a. residues that reveal the presence of the Dimerization Docking (D/D) domain, Catalytic interface domain and cAMP-Binding domain. The Chlamydomonas reinhardtii flagella harbour two AKAPs; viz., the radial spoke AKAP97 or RSP3 and the central pair AKAP240. Both these were identified on the basis of their RII-binding property. Interestingly, AKAP97 binds in vivo to two RII-like proteins (RSP7 and RSP11) that contain only the D/D domain. RESULTS: We found a Chlamydomonas Flagellar Associated Protein (FAP174) orthologous to MYCBP-1, a protein that binds to organellar AKAPs and Myc onco-protein. An in silico analysis shows that the N-terminus of FAP174 is similar to those RII domain-containing proteins that have binding affinities to AKAPs. Binding of FAP174 was tested with the AKAP97/RSP3 using in vitro pull down assays; however, this binding was rather poor with AKAP97/RSP3. Antibodies were generated against FAP174 and the cellular localization was studied using Western blotting and immunoflourescence in wild type and various flagella mutants. We show that FAP174 localises to the central pair of the axoneme. Using overlay assays we show that FAP174 binds AKAP240 previously identified in the C2 portion of the central pair apparatus. CONCLUSION: It appears that the flagella of Chlamydomonas reinhardtii contain proteins that bind to AKAPs and except for the D/D domain, lack the conventional a.a. stretches of PKA regulatory subunits (RSP7 and RSP11). We add FAP174 to this growing list.

Quantifying ligand-receptor interactions for gorge-spanning acetylcholinesterase inhibitors for the treatment of Alzheimer's disease.

There is a need for continued development of acetylcholinesterase (AChE) inhibitors that could prolong the life of acetylcholine in the synaptic cleft and also prevent the aggregation of amyloid peptides associated with Alzheimer's disease. The lack of a 3D-QSAR model which specifically deconvulates the type of interactions and quantifies them in terms of energies has motivated us to report a CoRIA model vis-à-vis the standard 3D-QSAR methods, CoMFA and CoMSIA. The CoRIA model was found to be statistically superior to the CoMFA and CoMSIA models and it could efficiently extract key residues involved in ligand recognition and binding to AChE. These interactions were quantified to gauge the magnitude of their contribution to the biological activity. In order to validate the CoRIA model, a pharmacophore map was first constructed and then used to virtually screen public databases, from which novel scaffolds were cherry picked that were not present in the training set. The biological activities of these novel molecules were then predicted by the CoRIA, CoMFA, and CoMSIA models. The hits identified were purchased and their biological activities were measured by the Ellman's method for AChE inhibition. The predicted activities are in unison with the experimentally measured biological activities.

Two calcium-dependent protein kinases from Chlamydomonas reinhardtii are transcriptionally regulated by nutrient starvation.

We report here, the transcriptional regulation of 2 Calcium Dependent Protein Kinases in response to nutrient starvation of Chlamydomonas reinhardtii vegetative cells. The CDPK proteins, CDPK1 and CDPK3; share 53% identity among themselves, a maximum of 57% and 52% to higher plants respectively and 42% to apicomplexan protozoans. We expressed a CDPK1-GFP fusion protein in the C. reinhardtii vegetative cells and showed its distribution both in the cell body and the membrane-matrix fraction of the flagella. The fusion protein exhibits mobility shift in the presence of Ca (2+), confirming its Ca (2+)-binding properties. To the best of our knowledge, this is the first report of transcriptional regulation of CDPKs from a unicellular chlorophyte in response to nutrient starvation namely acetate (A), phosphorus (P), and nitrogen (N).

Menadione-induced caspase-dependent programmed cell death in the green chlorophyte Chlamydomonas reinhardtii.

Menadione, a quinone that undergoes redox cycles leading to the formation of superoxide radicals, induces programmed cell death (PCD) in animals and plants. In this study, we investigated whether the unicellular green alga Chlamydomonas reinhardtii P.A.Dangeard is capable of executing PCD upon exposure to menadione stress. We report here, the morphological, molecular, and biochemical changes after menadione exposure of C. reinhardtii cells. The effect of menadione on cell death has been shown to be dose-dependent; 5-100 µM menadione causes 20%-46% cell death, respectively. It appears that growth is inhibited with the concomitant degradation of the photosynthetic pigments and by a decrease in the photosynthetic capacity. Being an oxidative stress, we found an H2 O2 burst within 15 min of menadione exposure, followed by an increase in antioxidant enzyme (superoxide dismutase [SOD], catalase [CAT], and ascorbate peroxidase [APX]) activities. In parallel, RT-PCR was performed for transcript analyses of Mn-SOD, CAT, and APX. Our results clearly revealed that expression of these genes were up-regulated upon menadione exposure. Furthermore, classical hallmarks of PCD such as alteration of mitochondrial membrane potential, significant increase in caspase-3-like DEVDase activity, cleavage of poly (ADP) ribose polymerase (PARP)-1-like enzyme, and DNA fragmentation as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay and oligosomal DNA fragmentation were observed. Moreover, antibodies against a mammalian active caspase-3 shared epitopes with a caspase-3-like protein of ~17 kDa; its pattern of expression and activity correlated with the onset of cell death. To the best of our knowledge, this is the first report on menadione-induced PCD through a mitochondrian-caspase protease pathway in an algal species.

Over-expression, purification and isotopic labeling of a tag-less human glucose-dependent insulinotropic polypeptide (hGIP).

Glucose-dependent insulinotropic polypeptide (GIP), a gut peptide released in response to food intake brings about secretion of insulin in a glucose-dependent manner upon binding to its receptor, GIPR. GIP-GIPR has emerged as a new vista for anti-diabetic drug discovery and their interaction is being probed at the atomic level to aid rational drug design. In order to probe this interaction on cells, the current study attempts towards expressing 15N-labeled GIP using classical molecular biology tools. We have developed a methodology to obtain GIP devoid of extra amino acid(s); a prerequisite to the intended interaction study. The synthetic GIP cDNA with a Factor Xa protease site at the N-terminus of GIP was inserted in the vector pET32a(+); the fusion protein thus expressed was eventually cleaved to obtain GIP. After successful Factor Xa cleavage, the cleaved GIP was confirmed by western blot. Subsequently, the (15N)GIP was obtained using the aforementioned procedure and confirmed by MALDI-TOF.

Studies on degradation efficiency of polycaprolactone by a naturally-occurring bacterium.

We report a strain of Bacillus, isolated from the rhizosphere of the mangrove Sesuvium portulacastrum, that degrades polycaprolactone (PCL) on timescales that are a factor of three shorter than hitherto reported, with complete degradation in only 20 days. The bacterium has been identified as Bacillus pumilus by means of 16S rRNA gene sequencing and FAME analysis; it secretes proteases and lipases and its 'de-polymerase' activity is evident by the zone of clearing in emulsified PCL. It is an aerobic chemoheterotroph capable of utilizing a variety of carbohydrates. Although not a true psychrophile, is a mesophile, growing optimally over a temperature range 30-45 degrees C and pH range 5-12.5. It is a halophile tolerating NaCI concentrations up to 10% w/v, and is unique in degrading and utilizing PCL and its monomer, epsilon-caprolactone (CL), as a sole carbon source. Degradation of PCL was monitored using Fourier Transform Infrared (FTIR) spectroscopy, light microscopy and scanning electron microscopy (SEM). This degradation was found to be enhanced by salts (NaCl, KCI, MgSO4, Na2HPO4) and at medium pH values in excess of 7. Under the same growth conditions, another standard Bacillus pumilus strain showed somewhat reduced PCL-degradation.