Role of Magmas in protein transport and human mitochondria biogenesis

Magmas, a conserved mammalian protein essential for eukaryotic development, is overexpressed in prostate carcinomas and cells exposed to granulocyte-macrophage colony-stimulating factor (GM-CSF). Reduced Magmas expression resulted in decreased proliferative rates in cultured cells. However, the cellular function of Magmas is still elusive. In this report, we have showed that human Magmas is an ortholog of Saccharomyces cerevisiae Pam16 having similar functions and is critical for protein translocation across mitochondrial inner membrane. Human Magmas shows a complete growth complementation of delta pam16 yeast cells at all temperatures. On the basis of our analysis, we report that Magmas localizes into mitochondria and is peripherally associated with inner mitochondrial membrane in yeast and humans. Magmas forms a stable subcomplex with J-protein Pam18 or DnaJC19 through its C-terminal region and is tethered to TIM23 complex of yeast and humans. Importantly, amino acid alterations in Magmas leads to reduced stability of the subcomplex with Pam18 that results in temperature sensitivity and in vivo protein translocation defects in yeast cells. These observations highlight the central role of Magmas in protein import and mitochondria biogenesis. In humans, absence of a functional DnaJC19 leads to dilated cardiac myophathic syndrome (DCM), a genetic disorder with characteristic features of cardiac myophathy and neurodegeneration. We propose that the mutations resulting in decreased stability of functional Magmas:DnaJC19 subcomplex at human TIM23 channel leads to impaired protein import and cellular respiration in DCM patients. Together, we propose a model showing how Magmas:DnaJC19 subcomplex is associated with TIM23 complex and thus regulates mitochondrial import process.

Control of morphogenesis in tobacco protoplast cultures: organogenesis vs embryogenesis

The morphogenetic pathway leading to plant differentiation in tobacco mesophyll protoplasts could be regulated. The course of development via organogenesis or embryogenesis was controlled by manipulating nutrient media, culture conditions and hormone requirements. A lowering of molarity of medium after 5 weeks of protoplast culture, inclusion of GA3 (0.5 mg/l) in the medium for first 8 weeks of culture and exclusion of reduced nitrogen in the medium resulted in shoot organogenesis, while maintenance of higher molarity of the medium till 8 weeks, reduced nitrogen in the medium and removal of 2, 4-D after 5 weeks of culture induced embryogenesis. Regenerability of viable plants was obtained by both developmental pathways. The implications of tobacco embryogenesis system in plant molecular genetics were highlighted.

The destabilization of human GCAP1 by a proline to leucine mutation might cause cone-rod dystrophy

Guanylate cyclase activating protein-1 (GCAP1) is required for activation of retinal guanylate cyclase-1 (RetGC1), which is essential for recovery of photoreceptor cells to the dark state. In this paper, experimentally derived observations are reported that help in explaining why a proline→leucine mutation at position 50 of human GCAP1 results in cone–rod dystrophy in a family carrying this mutation. The primary amino acid sequence of wild-type GCAP1 was mutated using site-directed mutagenesis to give a leucine at position 50. In addition, serine replaced a glutamic acid residue at position 6 to promote N‐terminal myristoylation, yielding the construct GCAP1 E6S/P50L. The enzyme was over-expressed in Escherichia coli cells, isolated and purified before being used in assays with RetGC1, characterized by circular dichroism (CD) spectroscopy, and investigated for protease resistance and thermal stability. Assays of cyclic guanosine monophosphate (cGMP) synthesis from guanosine triphosphate by RetGC1 in the presence of E6S/P50L showed that E6S/P50L could activate RetGC1 and displayed similar calcium sensitivity to wild-type GCAP1. In addition, E6S/P50L and wild-type GCAP1 possess similar CD spectra. However, there was a marked increase in the susceptibility to protease degradation and also a reduction in the thermal stability of E6S/P50L as observed by both the cGMP assay and CD spectroscopy. It is therefore suggested that although GCAP1 E6S/P50L has a similar activity and calcium dependency profile to the wild-type GCAP1, its lower stability could reduce its cellular concentration, which would in turn alter [Ca2+] and result in death of cells.

Enhanced J-protein interaction and compromised protein stability of mtHsp70 variants lead to mitochondrial dysfunction in Parkinsons disease

Parkinsons disease (PD) is the second most prevalent progressive neurological disorder commonly associated with impaired mitochondrial function in dopaminergic neurons. Although familial PD is multifactorial in nature, a recent genetic screen involving PD patients identified two mitochondrial Hsp70 variants (P509S and R126W) that are suggested in PD pathogenesis. However, molecular mechanisms underlying how mtHsp70 PD variants are centrally involved in PD progression is totally elusive. In this article, we provide mechanistic insights into the mitochondrial dysfunction associated with human mtHsp70 PD variants. Biochemically, the R126W variant showed severely compromised protein stability and was found highly susceptible to aggregation at physiological conditions. Strikingly, on the other hand, the P509S variant exhibits significantly enhanced interaction with J-protein cochaperones involved in folding and import machinery, thus altering the overall regulation of chaperone-mediated folding cycle and protein homeostasis. To assess the impact of mtHsp70 PD mutations at the cellular level, we developed yeast as a model system by making analogous mutations in Ssc1 ortholog. Interestingly, PD mutations in yeast (R103W and P486S) exhibit multiple in vivo phenotypes, which are associated with omitochondrial dysfunction', including compromised growth, impairment in protein translocation, reduced functional mitochondrial mass, mitochondrial DNA loss, respiratory incompetency and increased susceptibility to oxidative stress. In addition to that, R103W protein is prone to aggregate in vivo due to reduced stability, whereas P486S showed enhanced interaction with J-proteins, thus remarkably recapitulating the cellular defects that are observed in human PD variants. Taken together, our findings provide evidence in favor of direct involvement of mtHsp70 as a susceptibility factor in PD.

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.

Cellular and molecular regulation of mammalian blastocyst hatching

In mammals including humans, failure in blastocyst hatching and implantation leads to early embryonic loss and infertility. Prior to implantation, the blastocyst must hatch out of its acellular glycoprotein coat, the zona pellucida (ZP). The phenomenon of blastocyst hatching is believed to be regulated by (i) dynamic cellular components such as actin-based trophectodermal projections (TEPs), and (ii) a variety of autocrine and paracrine molecules such as growth factors, cytokines and proteases. The spatio-temporal regulation of zona lysis by blastocyst-derived cellular and molecular signaling factors is being keenly investigated. Our studies show that hamster blastocyst hatching is acelerated by growth factors such as heparin binding-epidermal growth factor and leukemia inhibitory factor and that embryo-derived, cysteine proteases including cathepsins are responsible for blastocyst hatching. Additionally, we believe that cyclooxygenase-generated prostaglandins, estradiol-17 beta mediated estrogen receptor-alpha signaling and possibly NF kappa B could be involved in peri-hatching development. Moreover, we show that TEPs are intimately involved with lysing ZP and that the TEPs potentially enrich and harbor hatching-enabling factors. These observations provide new insights into our understanding of the key cellular and molecular regulators involved in the phenomenon of mammalian blastocyst hatching, which is essential for the establishment of early pregnancy.

Insight into the early stages of thermal unfolding of peanut agglutinin by molecular dynamics simulations

Peanut agglutinin is a homotetrameric nonglycosylated protein. The protein has a unique open quaternary structure. Molecular dynamics simulations have been employed follow the atomistic details of its unfolding at different temperatures. The early events of the deoligomerization of the protein have been elucidated in the present study. Simulation trajectories of the monomer as well as those of the tetramer have been compared and the tetramer is found to be substantially more stable than its monomeric counterpart. The tetramer shows retention of most of its.. secondary structure but considerable loss of the tertiary structure at high temperature. e generation of a This observation impies the molten globule-like intermediate in the later stages of deoligomerization. The quaternary structure of the protein has weakened to a large extent, but none of the subunits are separated. In addition, the importance of the metal-binding to the stability of the protein structure has also been investigated. Binding of the metal ions not only enhances the local stability of the metal-ion binding loop, but also imparts a global stability to the overall structure. The dynamics of different interfaces vary significantly as probed through interface clusters. The differences are substantially enhanced at higher temperatures. The dynamics and the stability of the interfaces have been captured mainly by cluster analysis, which has provided detailed information on the thermal deoligomerization of the protein.

Inferring molecular function: contributions from functional linkages

In the current era of high-throughput sequencing and structure determination, functional annotation has become a bottleneck in biomedical science. Here, we show that automated inference of molecular function using functional linkages among genes increases the accuracy of functional assignments by >= 8% and enriches functional descriptions in >= 34% of top assignments. Furthermore, biochemical literature supports >80% of automated inferences for previously unannotated proteins. These results emphasize the benefit of incorporating functional linkages in protein annotation.

Crystal and molecular structure of the ammonium salt of the dinucleoside monophosphate d(CpG)

The crystal and molecular structure of the ammonium salt of deoxycytidylyl-(3'-5')-deoxyguanosine has been determined from 0.85 A resolution single crystal X-ray diffraction data. The crystals obtained by acetone diffusion technique at -20 degrees C, are orthorhombic, P212121, a = 12.880(2), b = 17444(2) and c = 27.642(2) A. The structure was solved by high resolution Patterson and Fourier methods and refined to R = 0.136. There are two d(CpG) molecules in the asymmetric unit forming a mini left handed Z-DNA helix. This is in contrast to the earlier reported forms of d(CpG) where the molecules form self base paired duplexes. There are two ammonium ions in the asymmetric unit. The major groove NH+4 ion interacts with N7 of guanines through water bridges besides making H-bonded interactions directly with the phosphate oxygen atoms. A second NH+4 ion is found in the minor groove interacting directly with the phosphate oxygen atoms. Symmetry related molecules pack in such a way that the cytosine base stacks on cytosine and guanine base on guanine. Our structure demonstrates that alternating d(CpG) sequences have the ability to adopt the left handed Z-DNA structure even at the dimer level i.e., in a sequence which is only two base pairs long.

Molecular systematics and conservation of the langurs and leaf monkeys of South Asia

Numerous morphology-based classification schemes have been proposed for langurs and leaf monkeys of South Asia but there is very little agreement between them. An incorrect classification scheme when used as a basis for biogeographic studies can support erroneous hypotheses. Further, lack of taxonomic resolution will also confound conservation efforts, given that conservation biologists use traditional morphology-based-classification schemes to prioritize species for conservation. Here, I have revisited recent molecular phylogenetic studies done on langurs and leaf monkeys of South Asia. Results from these studies are in turn used to derive a rational and scientific basis for prioritizing species for conservation. Molecular data support the classification of langurs of the Indian subcontinent-Hanuman, Nilgiri and purple-faced langurs-in the genus Semnopithecus, whereas Phayre's leaf monkey along with other Southeast Asian leaf monkeys form another distinct clade (Trachypithecus). The phylogenetic position of capped and golden langurs remains unresolved. Molecular data suggest that they are closely related to each other but this group might have evolved through past hybridization between Semnopithecus and Trachypithecus. Additionally, genetic data also support the splitting of the so-called Hanuman langurs into at least three species. The scores for taxonomic uniqueness of langurs and leaf monkeys of South Asia were revised using this molecular phylogeny-based classification. According to the revised scores, Phayres leaf monkey and golden langur are priority species for conservation followed by capped and Nilgiri langurs.

Studies on molecular evolution and structural features of double-headed inhibitors of ?-amylase and trypsin in plants

Plant seeds usually have high concentrations of proteinase and amylase inhibitors. These inhibitors exhibit a wide range of specificity, stability and oligomeric structure. In this communication, we report analysis of sequences that show statistically significant similarity to the double-headed alpha-amylase/trypsin inhibitor of ragi (Eleusine coracana). Our aim is to understand their evolutionary and structural features. The 14 sequences of this family that are available in the SWISSPROT database form three evolutionarily distinct branches. The branches relate to enzyme specificities and also probably to the oligomeric state of the proteins and not to the botanical class of the plant from which the enzymes are derived. This suggests that the enzyme specificities of the inhibitors evolved before the divergence of commercially cultivated cereals. The inhibitor sequences have three regions that display periodicity in hydrophobicity. It is likely that this feature reflects extended secondary structure in these segments. One of the most variable regions of the polypeptide corresponds to a loop, which is most probably exposed in the native structure of the inhibitors and is responsible for the inhibitory property.

Molecular organization of the gene encoding Xenopus laevis transforming growth factor-beta 5

Transforming growth factor-beta s (TGF-beta 5) are multifunctional polypeptides, known to influence proliferation and differentiation of many cell types. TGF-beta 5 cDNA was cloned from Xenopus laevis and this isoform is unique to the amphibians. Here, we report the isolation and characterization of the TGF-beta 5 genomic clones to determine the structure of TGF-beta 5 gene. The gene consists of seven exons and all intron-exon boundaries follow the GT-AG consensus. The organization of TGF-beta 5 gene was identical to that of the mammalian TGF-beta isoforms, with the exception of position of the first splice junction. We determined the size of TGF-beta 5 gene to be approximately 20 kb.

The genetics of Mycobacterium tuberculosis

Gene manipulation in Mycobacterium tuberculosis has been slow in coming of age owing to the inherent difficulties associated with working on this aerosol-transmitted pathogen, in addition to the paucity of molecular tools such as plasmids and transposons. One of the early approaches to overcome these difficulties was the development of phasmids, which combined the properties of phages and plasmids and allowed introduction of recombinant genes into mycobacteria. The lone plasmid pAL5000 of mycobacteria has been exploited to its fullest potential in the construction of a plethora of vectors. Above all, the single most important achievement has been the development of elegant and innovative approaches to overcome the problem of illegitimate recombination which threatened the success of allelic-exchange mutagenesis in the slow-growing pathogenic mycobacterial species. In this review I discuss the current status of conditionally replicating plasmid and transposon vectors and their application in generating targeted mutations in mycobacteria.