FAIR: A server for internal sequence repeats

An Internet computing server has been developed to identify all the occurrences of the internal sequence repeats in a protein and DNA sequences. Further, an option is provided for the users to check the occurrence(s) of the resultant sequence repeats in the other sequence and structure (Protein Data Bank) databases. The databases deployed in the proposed computing engine are up-to-date and thus the users will get the latest information available in the respective databases. The server is freely accessible over the World Wide Web (WWW). AVAILABILITY: http://bioserver1.physics.iisc.ernet.in/fair/     

PDK1 Recruitment to the SHPS-1 Signaling Complex Enhances Insulin-like Growth Factor-I-stimulated AKT Activation and Vascular Smooth Muscle Cell Survival

In vascular smooth muscle cells, exposed to hyperglycemia and insulin-like growth factor-I (IGF-I), SHPS-1 functions as a scaffold protein, and a signaling complex is assembled that leads to AKT activation. However, the underlying mechanism by which formation of this complex activates the kinase that phosphorylates AKT (Thr³⁰⁸) is unknown. Therefore, we investigated the mechanism of PDK1 recruitment to the SHPS-1 signaling complex and the consequences of disrupting PDK1 recruitment for downstream signaling. Our results show that following IGF-I stimulation, PDK1 is recruited to SHPS-1, and its recruitment is mediated by Grb2, which associates with SHPS-1 via its interaction with Pyk2, a component of the SHPS-1-associated complex. A proline-rich sequence in PDK1 bound to an Src homology 3 domain in Grb2 in response to IGF-I. Disruption of Grb2-PDK1 by expression of either a Grb2 Src homology 3 domain or a PDK1 proline to alanine mutant inhibited PDK1 recruitment to SHPS-1, leading to impaired IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation. Following its recruitment to SHPS-1, PDK1 was further activated via Tyr^373/376 phosphorylation, and this was required for a maximal increase in PDK1 kinase activity and AKT-mediated FOXO3a Thr³² phosphorylation. PDK1 recruitment was also required for IGF-I to prevent apoptosis that occurred in response to hyperglycemia. Assembly of the Grb2-PDK1 complex on SHPS-1 was specific for IGF-I signaling because inhibiting PDK1 recruitment to SHPS-1 had no effect on EGF-stimulated AKT Thr³⁰⁸ phosphorylation. These findings reveal a novel mechanism for recruitment of PDK1 to the SHPS-1 signaling complex, which is required for IGF-I-stimulated AKT Thr³⁰⁸ phosphorylation and inhibition of apoptosis.     

Human RECQ5beta helicase promotes strand exchange on synthetic DNA structures resembling a stalled replication fork

The role of the human RECQ5β helicase in the maintenance of genomic stability remains elusive. Here we show that RECQ5β promotes strand exchange between arms of synthetic forked DNA structures resembling a stalled replication fork in a reaction dependent on ATP hydrolysis. BLM and WRN can also promote strand exchange on these structures. However, in the presence of human replication protein A (hRPA), the action of these RecQ-type helicases is strongly biased towards unwinding of the parental duplex, an effect not seen with RECQ5β. A domain within the non-conserved portion of RECQ5β is identified as being important for its ability to unwind the lagging-strand arm and to promote strand exchange on hRPA-coated forked structures. We also show that RECQ5β associates with DNA replication factories in S phase nuclei and persists at the sites of stalled replication forks after exposure of cells to UV irradiation. Moreover, RECQ5β is found to physically interact with the polymerase processivity factor proliferating cell nuclear antigen in vitro and in vivo. Collectively, these findings suggest that RECQ5β may promote regression of stalled replication forks to facilitate the bypass of replication-blocking lesions by template-switching. Loss of such activity could explain the elevated level of mitotic crossovers observed in RECQ5β-deficient cells.     

Integration of a physiologically-based pharmacokinetic model with a whole-body,organ-resolved genome-scale model for characterization of ethanol and acetaldehyde metabolism

Ethanol is one of the most widely used recreational substances in the world and due to its ubiquitous use, ethanol abuse has been the cause of over 3.3 million deaths each year. In addition to its effects, ethanol’s primary metabolite, acetaldehyde, is a carcinogen that can cause symptoms of facial flushing, headaches, and nausea. How strongly ethanol or acetaldehyde affects an individual depends highly on the genetic polymorphisms of certain genes. In particular, the genetic polymorphisms of mitochondrial aldehyde dehydrogenase, ALDH2, play a large role in the metabolism of acetaldehyde. Thus, it is important to characterize how genetic variations can lead to different exposures and responses to ethanol and acetaldehyde. While the pharmacokinetics of ethanol metabolism through alcohol dehydrogenase have been thoroughly explored in previous studies, in this paper, we combined a base physiologically-based pharmacokinetic (PBPK) model with a whole-body genome-scale model (WBM) to gain further insight into the effect of other less explored processes and genetic variations on ethanol metabolism. This combined model was fit to clinical data and used to show the effect of alcohol concentrations, organ damage, ALDH2 enzyme polymorphisms, and ALDH2-inhibiting drug disulfiram on ethanol and acetaldehyde exposure. Through estimating the reaction rates of auxiliary processes with dynamic Flux Balance Analysis, The PBPK-WBM was able to navigate around a lack of kinetic constants traditionally associated with PK modelling and demonstrate the compensatory effects of the body in response to decreased liver enzyme expression. Additionally, the model demonstrated that acetaldehyde exposure increased with higher dosages of disulfiram and decreased ALDH2 efficiency, and that moderate consumption rates of ethanol could lead to unexpected accumulations in acetaldehyde. This modelling framework combines the comprehensive steady-state analyses from genome-scale models with the dynamics of traditional PK models to create a highly personalized form of PBPK modelling that can push the boundaries of precision medicine.     

Pro-apoptotic role of NF-kappaB: implications for cancer therapy

Nuclear factor-kappaB (NF-kappaB) is generally viewed as anti-apoptotic and oncogenic, leading to a quest for its inhibitors. However, recent evidence suggests that in some situations NF-kappaB may promote apoptosis. Depending on the specific cell type and the stimulus involved, NF-kappaB activation may lead to either anti- or pro-apoptotic response. Both these effects can be mediated by NF-kappaB in a context-dependent manner by selectively regulating its target genes. In this review, we discuss the evidence for NF-kappaB's pro-apoptotic role and explore the possible mechanisms behind it. We emphasize that rather than trying to inhibit NF-kappaB in cancer therapy, agents should be developed to unleash its pro-apoptotic ability.     

Comparative investigation of the differentiation capability of bone-marrow- and adipose-derived mesenchymal stem cells by qualitative and quantitative analysis

Mesenchymal stem cells (MSCs) hold promise for cell-based therapy in regenerative medicine. To date, MSCs have been obtained from conventional bone marrow via a highly invasive procedure. Therefore, MSCs are now also isolated from sources such as adipose tissue, cord blood and cord stroma, a subject of growing interest. As the characterization and differentiation potential of adipose-derived MSCs (AD-MSCs) and bone-marrow-derived MSCs (BM-MSCs) have not been documented, we have evaluated and compared the characteristics of both MSC types by qualitative and quantitative analyses. Both cell types show similar morphology and surface protein expression, being positive for stromal-associated markers and negative for hematopoietic and endothelial markers. The colony-forming potential of AD-MSCs is distinctly higher than that of BM-MSCs. Nonetheless, similar adipogenic and osteogenic differentiation is observed in both groups of MSCs. Cytochemical qualitative analysis and calcium mineralization demonstrate higher levels toward osteogenic differentiation in BM-MSCs than in AD-MSCs. On the contrary, the percentage of Nile red oil staining for differentiated adipocytes is higher in AD-MSCs than in BM-MSCs. Quantitative real-time polymerase chain reaction shows similar patterns of osteogenic- and adipogenic-associated gene expression in both cell types. Each of the MSCs respond in functional analysis by exhibiting unique properties at the differentiation level according to their micro-environmental niche. Thus, quantitative analysis might be a valuable means of describing stem cell multipotency, in addition to qualitative investigation.     

The non-catalytic N-terminal extension of formylglycine-generating enzyme is required for its biological activity and retention in the endoplasmic reticulum

Formylglycine-generating enzyme (FGE) catalyzes the oxidation of a specific cysteine residue in nascent sulfatase polypeptides to formylglycine (FGly). This FGly is part of the active site of all sulfatases and is required for their catalytic activity. Here we demonstrate that residues 34-68 constitute an N-terminal extension of the FGE catalytic core that is dispensable for in vitro enzymatic activity of FGE but is required for its in vivo activity in the endoplasmic reticulum (ER), i.e. for generation of FGly residues in nascent sulfatases. In addition, this extension is needed for the retention of FGE in the ER. Fusing a KDEL retention signal to the C terminus of FGE is sufficient to mediate retention of an N-terminally truncated FGE but not sufficient to restore its biological activity. Fusion of FGE residues 1-88 to secretory proteins resulted in ER retention of the fusion protein. Moreover, when fused to the paralog of FGE (pFGE), which itself lacks FGly-generating activity, the FGE extension (residues 34-88) of this hybrid construct led to partial restoration of the biological activity of co-expressed N-terminally truncated FGE. Within the FGE N-terminal extension cysteine 52 is critical for the biological activity. We postulate that this N-terminal region of FGE mediates the interaction with an ER component to be identified and that this interaction is required for both the generation of FGly residues in nascent sulfatase polypeptides and for retention of FGE in the ER.     

Program review. Challenges and opportunities for training the next generation of biophysicists: perspectives of the directors of the Molecular Biophysics Training Program at Northwestern University

Molecular biophysics is a broad, diverse, and dynamic field that has presented a variety of unique challenges and opportunities for training future generations of investigators. Having been or currently being intimately associated with the Molecular Biophysics Training Program at Northwestern, we present our perspectives on various issues that we have encountered over the years. We propose no cookie-cutter solutions, as there is no consensus on what constitutes the "ideal" program. However, there is uniformity in opinion on some key issues that might be useful to those interested in establishing a biophysics training program.