Interaction of discoidin domain receptor 1 with collagen type 1

Discoidin domain receptor 1 (DDR1) is a widely expressed tyrosine kinase receptor which binds to and gets activated by collagens including collagen type 1. Little is understood about the interaction of DDR1 with collagen and its possible functional implications. Here, we elucidate the binding pattern of the DDR1 extracellular domain (ECD) to collagen type 1 and its impact on collagen fibrillogenesis. Our in vitro assays utilized DDR1-Fc fusion proteins, which contain only the ECD of DDR1. Using surface plasmon resonance, we confirmed that further oligomerization of DDR1-Fc (by means of anti-Fc antibody) greatly enhances its binding to immobilized collagen type 1. Single-molecule imaging by means of atomic force microscopy revealed that DDR1 oligomers bound at overlapping or adjacent collagen molecules and were nearly absent on isolated collagen molecules. Interaction of DDR1 oligomers with collagen was found to modulate collagen fibrillogenesis both in vitro and in cell-based assays. Collagen fibers formed in the presence of DDR1 had a larger average diameter, were more cross-linked and lacked the native banded structure. The presence of DDR1 ECD resulted in "locking" of collagen molecules in an incomplete fibrillar state both in vitro and on surfaces of cells overexpressing DDR1. Our results signify an important functional role of the DDR1 ECD, which occurs naturally in kinase-dead isoforms of DDR1 and as a shedded soluble protein. The modulation of collagen fibrillogenesis by the DDR1 ECD elucidates a novel mechanism of collagen regulation by DDR1.     

Necessary and sufficient factors for the import of transfer RNA into the kinetoplast mitochondrion

The mechanism of active transport of transfer RNA (tRNA) across membranes is largely unknown. Factors mediating the import of tRNA into the kinetoplast mitochondrion of the protozoon Leishmania tropica are organized into a multiprotein RNA import complex (RIC) at the inner membrane. Here, we present the complete characterization of the identities and functions of the subunits of this complex. The complex contains three mitochondrion- and eight nuclear-encoded subunits; six of the latter are necessary and sufficient for import. Antisense-mediated knockdown of essential subunits resulted in the depletion of mitochondrial tRNAs and inhibition of organellar translation. Functional complexes were reconstituted with recombinant subunits expressed in Escherichia coli. Several essential RIC subunits are identical to specific subunits of respiratory complexes. These findings provide new information on the evolution of tRNA import and the foundation for detailed structural and mechanistic studies.     

The DNA Replication,Repair, and Recombination Pathway Genes Modulating Yield and Stress Tolerance Traits in Chickpea

DNA replication, repair, and recombination (DRRR) are the fundamental processes required for faithful transmission of genetic information within and between generations. The DRRR genes protect the cells from potential mutations and damage during the developmental phases and stress conditions. Thus, these genes indirectly regulate diverse important agronomic traits in a crop plant. A genome-wide survey of six DRRR pathway genes, namely, DNA replication, Base Excision Repair, Nucleotide Excision Repair, Homologous Recombination, Mismatch Excision Repair, and Non-Homologous End-Joining, identified 157 DRRR genes in chickpea. Phylogenetic analysis of these genes within the legume clades and model plant Arabidopsis identified 42 conserved DRRR genes exhibiting clade-specific evolutionary patterns. Integrating the gene-based association mapping with differential expression profiling identified the natural alleles of the potential DRRR genes, primarily regulating flowering and maturation time and involved in drought tolerance of chickpea. Identifying and understanding DRRR genes’ roles in regulating yield and stress tolerance traits in a vital grain legume like chickpea is requisite for its future crop improvement endeavors. Manipulation of promising functionally relevant DRRR genes will pave the way for simultaneous improvement in multiple beneficial agronomic traits in chickpea.

     

Vascular gene polymorphisms (EDNRA -231 G>A and APOE HhaI) and risk for migraine

Migraine is a neurovascular disorder, and hence, any alteration in vascular endothelial function by either the endothelin system or the apolipoproteins may contribute to its pathophysiology. Thus, we investigated the role of EDNRA -231 G>A and APOE HhaI polymorphism for a possible association with migraine. Genotyping of 613 subjects consisting of 217 migraine subjects, 217 healthy controls (HC), and 179 subjects with tension-type headache was performed using the standard PCR-RFLP method. Data were analyzed by taking the Bonferroni-corrected p-values into account. We found significant difference in the frequency of EDNRA AA genotype between migraine subjects when compared with HC (p-value?=?0.005; OR?=?2.542; confidence interval [CI]?=?1.329-4.863). A similar trend was shown by female migraine subjects at genotype and allele levels. The association of EDNRA -231 G>A polymorphism with migraine fit a recessive model (migraine vs. HC, p-value?=?0.002; OR?=?1.917; CI?=?2.268-2.898). Female migraineurs without aura (MO) followed a similar trend. In the case of APOE HhaI polymorphism, E3E4 and E2E3 genotypes conferred risk when taken together in case of migraine versus HC (p-value?=?0.005; OR?=?2.715; CI?=?1.342-5.490) and migraine with aura (MA) versus HC (p-value?=?0.004; OR?=?3.422; CI?=?7.992). The risk was also seen after stratification on the basis of gender in female migraineurs (total migraine and MA). The interaction of EDNRA and APOE genotypes did not show further significance. The AA genotype and A allele of EDNRA -231 G>A polymorphism conferred risk for total migraine and MO. In APOE HhaI polymorphism, E3E4 and E2E3 conferred risk when taken together in total migraine and MA.     

Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB

Background

The integrated functions of 11 Ser/Thr protein kinases (STPKs) and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP) is regulated through phosphorylation by STPKs.

Principal Findings

PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn²⁺-ions and inorganic phosphate (Pi). PstP is differentially phosphorylated on the cytosolic domain with Thr¹³⁷, Thr¹⁴¹, Thr¹⁷⁴ and Thr²⁹⁰ being the target residues of PknB while Thr¹³⁷ and Thr¹⁷⁴ are phosphorylated by PknA. The Mn²⁺-ion binding residues Asp³⁸ and Asp²²⁹ are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP_c ^D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn²⁺/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.

Conclusions and Significance

This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.     

Regulation of histone synthesis and nucleosome assembly

Histone deposition onto nascent DNA is the first step in the process of chromatin assembly during DNA replication. The process of nucleosome assembly represents a daunting task for S-phase cells, partly because cells need to rapidly package nascent DNA into nucleosomes while avoiding the generation of excess histones. Consequently, cells have evolved a number of nucleosome assembly factors and regulatory mechanisms that collectively function to coordinate the rates of histone and DNA synthesis during both normal cell cycle progression and in response to conditions that interfere with DNA replication.