Spectral plots and the representation and interpretation of biological data

It is basic question in biology and other fields to identify the characteristic properties that on one hand are shared by structures from a particular realm, like gene regulation, protein–protein interaction or neural networks or foodwebs, and that on the other hand distinguish them from other structures. We introduce and apply a general method, based on the spectrum of the normalized graph Laplacian, that yields representations, the spectral plots, that allow us to find and visualize such properties systematically. We present such visualizations for a wide range of biological networks and compare them with those for networks derived from theoretical schemes. The differences that we find are quite striking and suggest that the search for universal properties of biological networks should be complemented by an understanding of more specific features of biological organization principles at different scales.

The Role of the Mammalian DNA End-processing Enzyme Polynucleotide Kinase 3’-Phosphatase in Spinocerebellar Ataxia Type 3 Pathogenesis

DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3’-P and 5’-OH, are processed by mammalian polynucleotide kinase 3’-phosphatase (PNKP), a bifunctional enzyme with 3’-phosphatase and 5’-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14–41 to 55–82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP’s 3’ phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3’-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients’ brain. Finally, long amplicon quantitative PCR analysis of human MJD patients’ brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.     

A novel meta-cleavage product hydrolase from Flavobacterium sp. ATCC27551

The organophosphate degrading (opd) gene cluster of plasmid pPDL2 of Flavobacterium sp. ATCC27551 contains a novel open-reading frame, orf243. This was predicted to encode an alpha/beta hydrolase distantly related to the meta-fission product (MFP) hydrolases such as XylF, PhnD, and CumD. By homology modeling Orf243 has most of the structural features of MFP hydrolases including the characteristic active site catalytic triad. The purified protein (designated MfhA) is a homotetramer and shows similar affinity for 2-hydroxy-6-oxohepta-2,4-dienoate (HOHD), 2-hydroxymuconic semialdehyde (HMSA), and 2-hydroxy-5-methylmuconic semialdehyde (HMMSA), the meta-fission products of 3-methyl catechol, catechol, and 4-methyl catechol. The unique catalytic properties of MfhA and the presence near its structural gene of cis-elements required for transposition suggest that mfhA has evolved towards encoding a common hydrolase that can act on meta-fission products containing either aldehyde or ketone groups.     

Taxoid from the needles of the Himalayan yew Taxus wallichiana with cytotoxic and immunomodulatory activities

The needles of Taxus wallichiana gave a taxoid 1-hydroxy- 2-deacetoxy-5-decinnamoyl-taxinine j, whose structure has been established by spectroscopic data and confirmed by X-ray crystallography. The taxoid possesses significant cytotoxic and immunomodulatory activity.     

Lipopolysaccharide bound structures of the active fragments of fowlicidin-1, a cathelicidin family of antimicrobial and antiendotoxic peptide from chicken, determined by transferred nuclear Overhauser effect spectroscopy

Cathelicidins comprise a major family of host-defense antimicrobial peptides in vertebrates. The C-terminal part of the cathelicidins is bestowed with antimicrobial and lipopolysaccharide (LPS) neutralizing activities. In this work, we repot high resolution solution structures of two nontoxic active fragments, residues 1-16 or RG16 and residues 8-26 or LK19, of fowlicidin-1, a cathelicidin family of peptide from chicken, as a complex with LPS using two-dimensional transferred nuclear Overhauser effect (Tr-NOE) spectroscopy. Both peptides are highly flexible and do not assume any preferred conformations in their free states. Upon complexation with endotoxin or LPS, peptides undergo structural transitions towards folded conformations. Structure calculations reveal that the LK19 peptide adopts a well defined helical structure with a bend at the middle. By contrast, the first seven amino acids of RG16 are found to be flexible followed by a helical conformation for the residues L8-A15. In addition, a truncated version of LK19 encompassing residues A15-K26 or AK12 displays an amphipathic helical structure in LPS. Saturation transfer difference (STD) NMR studies demonstrate that all peptides, RG16, LK19, and AK12, are in close proximity with LPS, whereby the aromatic residues showed the strongest STD effects. Fluorescence studies with fluorescein isothiocyanate (FITC) labeled LPS in the presence of full-length fowlicidin-1, LK19, RG16, and AK12 indicated that LPS-neutralization property of these peptides may result from plausible dissociation of LPS aggregates. The helical structures of peptide fragments derived from fowlicidin-1 in LPS could be utilized to develop nontoxic antiendotoxic compounds.     

Enantioselective Nitrilase from <Emphasis Type="Italic">Pseudomonas putida</Emphasis>: Cloning,Heterologous Expression,and Bioreactor Studies

Nitrilases have attracted tremendous attention for the preparation of optically pure carboxylic acids. This article aims to address the production and utilization of a highly enantioselective nitrilase from Pseudomonas putida MTCC 5110 for the hydrolysis of racemic mandelonitrile to (R)-mandelic acid. The nitrilase gene from P. putida was cloned in pET 21b(+) and over-expressed as histidine-tagged protein in Escherichia coli. The histidine-tagged enzyme was purified from crude cell extracts of IPTG-induced cells of E. coli BL21 (DE3). Inducer replacement studies led to the identification of lactose as a suitable and cheap alternative to the costly IPTG. Effects of medium components, various physico-chemical, and process parameters (pH, temperature, aeration, and agitation) for the production of nitrilase by engineered E. coli were optimized and scaled up to a laboratory scale bioreactor (6.6 l). Finally, the recombinant E. coli whole-cells were utilized for the production of (R)-(−)-mandelic acid.     

Evidence for a “Wattle and Daub” Model of the Cyst Wall of Entamoeba

The cyst wall of Entamoeba invadens (Ei), a model for the human pathogen Entamoeba histolytica, is composed of fibrils of chitin and three chitin-binding lectins called Jacob, Jessie3, and chitinase. Here we show chitin, which was detected with wheat germ agglutinin, is made in secretory vesicles prior to its deposition on the surface of encysting Ei. Jacob lectins, which have tandemly arrayed chitin-binding domains (CBDs), and chitinase, which has an N-terminal CBD, were each made early during encystation. These results are consistent with their hypothesized roles in cross-linking chitin fibrils (Jacob lectins) and remodeling the cyst wall (chitinase). Jessie3 lectins likely form the mortar or daub of the cyst wall, because 1) Jessie lectins were made late during encystation; 2) the addition to Jessie lectins to the cyst wall correlated with a marked decrease in the permeability of cysts to nucleic acid stains (DAPI) and actin-binding heptapeptide (phalloidin); and 3) recombinant Jessie lectins, expressed as a maltose-binding proteins in the periplasm of Escherichia coli, caused transformed bacteria to agglutinate in suspension and form a hard pellet that did not dissociate after centrifugation. Jessie3 appeared as linear forms and rosettes by negative staining of secreted recombinant proteins. These findings provide evidence for a “wattle and daub” model of the Entamoeba cyst wall, where the wattle or sticks (chitin fibrils likely cross-linked by Jacob lectins) is constructed prior to the addition of the mortar or daub (Jessie3 lectins).     

S-acylation of SARS-CoV-2 spike protein: Mechanistic dissection,in vitro reconstitution and role in viral infectivity

S-acylation, also known as palmitoylation, is the most widely prevalent form of protein lipidation, whereby long-chain fatty acids get attached to cysteine residues facing the cytosol. In humans, 23 members of the zDHHC family of integral membrane enzymes catalyze this modification. S-acylation is critical for the life cycle of many enveloped viruses. The Spike protein of SARS-CoV-2, the causative agent of COVID-19, has the most cysteine-rich cytoplasmic tail among known human pathogens in the closely related family of β-coronaviruses; however, it is unclear which of the cytoplasmic cysteines are S-acylated, and what the impact of this modification is on viral infectivity. Here we identify specific cysteine clusters in the Spike protein of SARS-CoV-2 that are targets of S-acylation. Interestingly, when we investigated the effect of the cysteine clusters using pseudotyped virus, mutation of the same three clusters of cysteines severely compromised viral infectivity. We developed a library of expression constructs of human zDHHC enzymes and used them to identify zDHHC enzymes that can S-acylate SARS-CoV-2 Spike protein. Finally, we reconstituted S-acylation of SARS-CoV-2 Spike protein in vitro using purified zDHHC enzymes. We observe a striking heterogeneity in the S-acylation status of the different cysteines in our in cellulo experiments, which, remarkably, was recapitulated by the in vitro assay. Altogether, these results bolster our understanding of a poorly understood posttranslational modification integral to the SARS-CoV-2 Spike protein. This study opens up avenues for further mechanistic dissection and lays the groundwork toward developing future strategies that could aid in the identification of targeted small-molecule modulators.     

GC-rich intra-operonic spacers in prokaryotes: possible relation to gene order conservation

Genome-scale compositional analyses of non-coding sequences from 410 microbes of varying GC-content, lineage, environment/life-style, reveal presence of a distinct trend in GC-usage in spacers between intra-operonic and extra-operonic gene-pairs. For most of the microbes, average GC-content of the intra-operonic spacers are consistently higher than those between extra-operonic unidirectional gene-pairs. Also, unidirectional gene-pairs exhibiting higher cross-species conservation, irrespective of their operonic context, house relatively GC-rich spacers. A few prokaryotes, most of which represent known cases of genome degradation, stand out as exceptions defying this trend. GC-enrichment of intra-operonic spacers therefore appears to be an evolutionary strategy facilitating preservation of operonic gene-order.