Using diffusion distances for flexible molecular shape comparison

Background  

Many molecules are flexible and undergo significant shape deformation as part of their function, and yet most existing molecular shape comparison (MSC) methods treat them as rigid bodies, which may lead to incorrect shape recognition.     

The role of integration host factor In gene expression in Escherichia coli

Integration host factor is a sequence-specific, histone-like, multifunctional DNA-binding and -bending protein of Escherichia coli. The characterization and functional analysis of this protein has been done mainly in bacteriophage lambda and other mobile genetic elements. Less is known concerning the role of integration host factor (IHF) in E. coli, although it has been implicated in a number of processes in this organism including DNA replication, site-specific recombination, and gene expression. This review presents recent work which suggests that IHF alters the activity of an unusually large number of operons in E. coli. We discuss the possible physiological relevance of the involvement of IHF in gene expression and the hypothesis that IHF is a member of a class of functionally redundant proteins that participate in chromosome structure and multiple processes involving DNA.     

Integration host factor amplifies the induction of the aceBAK operon of Escherichia coli by relieving IclR repression.

A binding site for integration host factor (IHF) was identified upstream of the aceBAK promoter. Under inducing conditions, IHF activates aceB::lacZ expression by opposing IclR repression. In contrast, IHF has little effect on aceB::lacZ expression under repressing conditions. The ability of IHF to relieve repression under inducing but not repressing conditions allows this protein to amplify the induction of aceBAK.     

“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

Quantitative assessment of hemoglobin-induced endothelial barrier dysfunction.

Hemoglobin (Hb)-based O2 carriers (HBOC) are undergoing extensive development as potential "blood substitutes." A major problem associated with these molecules is an increase in microvascular permeability and peripheral vascular resistance. In this paper, we utilized bovine lung microvascular endothelial cell monolayers and simultaneously measured Hb-induced changes in transendothelial electrical resistance, diffusive albumin permeability, and diffusive Hb permeability (PDH) for three forms of Hb: natural tetrameric human Hb-A and two polymerized recombinant HBOCs containing alpha-human and beta-bovine chains designated Hb-Polytaur (molecular mass: 500 kDa) and Hb-(Polytaur)n (molecular mass: approximately 1,000,000 Da), respectively. Hb-Polytaur and Hb-(Polytaur)n are being evaluated for clinical use as HBOCs. All three Hb molecules induce a rapid decline of transendothelial electrical resistance to 30% of baseline. Diffusive albumin permeabiltiy increases, on average, approximately ninefold (2.78 x 10(-7) vs. 2.47 x 10(-6) cm/s) in response to Hb exposure. All three Hb molecules induce an increase in their own permeability, a process that we have called Hb-induced Hb permeability. The magnitude of change of PDH is also related to Hb size. When PDH is corrected for the diffusive coefficient for each Hb species, no evidence of restricted diffusion is found. Immunofluorescent images demonstrate Hb-induced actin stress fiber formation and large intercellular gaps. These data provide the first quantitative assessment of the effect of polymerized HBOC on their own diffusion rates over time. We discuss the importance of these findings in terms of Hb extravasation rates, molecular sieving, and clinical consequences of HBOC use.     

Accumulation of LEA proteins in salt (NaCl) stressed young seedlings of rice (Oryza sativa L.) cultivar Bura Rata and their degradation during recovery from salinity stress

Germination and subsequent hydroponic growth under salt stress (100 mmol/L NaCl) triggered an accumulation of six major stress proteins and resulted in a growth arrest of young seedlings of rice (Oryza sativa L.) cv. Bura Rata. Based on two-dimensional electrophoretic resolution, partial amino acid sequencing and immunodetection techniques, four of the salt stress-induced polypeptides were identified as LEA proteins. Under all experimental conditions wherein seedlings exhibited superior halotolerance, salt stress-induced LEA proteins were expressed at low levels. In contrast, accumulation of LEA proteins was found associated with growth arrest. When returned to non-saline media, seedlings stressed with salt for four days recovered immediately. Longer exposure to 100 mmol/L NaCl, however, progressively delayed recovery and reduced the number of seedlings which could recover from salt stress. Recovery from salt stress was consistently accompanied by degradation of the salt stress-induced LEA proteins. The results of this study show that LEA proteins accumulate during the salinity-triggered growth arrest of young Bura Rata seedlings and are mobilised during the recovery of seedlings from salinity stress.     

Canstatin, a novel matrix-derived inhibitor of angiogenesis and tumor growth

We isolated and identified an endogenous 24-kDa human basement membrane-derived inhibitor of angiogenesis and tumor growth, termed canstatin. Canstatin, a fragment of the alpha2 chain of type IV collagen, was produced as a recombinant molecule in Escherichia coli and 293 embryonic kidneys cells. Canstatin significantly inhibited human endothelial cell migration and murine endothelial cell tube formation. Additionally, canstatin potently inhibited 10% fetal bovine serum-stimulated endothelial cell proliferation and induced apoptosis, with no inhibition of proliferation or apoptosis observed on non-endothelial cells. Inhibition of endothelial proliferation was not concomitant with a change in extracellular signal-regulated kinase activation. We demonstrate that apoptosis induced by canstatin was associated with a down-regulation of the anti-apoptotic protein, FLIP. Canstatin also suppressed in vivo growth of large and small size tumors in two human xenograft mouse models with histology revealing decreased CD31-positive vasculature. Collectively, these results suggest that canstatin is a powerful therapeutic molecule for suppressing angiogenesis.     

Compound Heterozygosity for Y Box Proteins Causes Sterility Due to Loss of Translational Repression

The Y-box proteins YBX2 and YBX3 bind RNA and DNA and are required for metazoan development and fertility. However, possible functional redundancy between YBX2 and YBX3 has prevented elucidation of their molecular function as RNA masking proteins and identification of their target RNAs. To investigate possible functional redundancy between YBX2 and YBX3, we attempted to construct Ybx2 ^-/- ;Ybx3 ^-/- double mutants using a previously reported Ybx2 ^-/- model and a newly generated global Ybx3 ^-/- model. Loss of YBX3 resulted in reduced male fertility and defects in spermatid differentiation. However, homozygous double mutants could not be generated as haploinsufficiency of both Ybx2 and Ybx3 caused sterility characterized by extensive defects in spermatid differentiation. RNA sequence analysis of mRNP and polysome occupancy in single and compound Ybx2/3 heterozygotes revealed loss of translational repression almost exclusively in the compound Ybx2/3 heterozygotes. RNAseq analysis also demonstrated that Y-box protein dose-dependent loss of translational regulation was inversely correlated with the presence of a Y box recognition target sequence, suggesting that Y box proteins bind RNA hierarchically to modulate translation in a range of targets.