Faculty Bibliography

Endoplasmic reticulum (ER) homeostasis requires transfer and subsequent processing of the glycan Glc(3)Man(9)GlcNAc(2) (G(3)M(9)Gn(2)) from the lipid-linked oligosaccharide (LLO) glucose(3)mannose(9)N-acetylglucosamine(2)-P-P-dolichol (G(3)M(9)Gn(2)-P-P-Dol) to asparaginyl residues of nascent glycoprotein precursor polypeptides. However, it is unclear how the ER is protected against dysfunction from abnormal accumulation of LLO intermediates and aberrant N-glycosylation, as occurs in certain metabolic diseases. In metazoans phosphorylation of eukaryotic initiation factor 2alpha (eIF2alpha) on Ser(51) by PERK (PKR-like ER kinase), which is activated by ER stress, attenuates translation initiation. We use brief glucose deprivation to simulate LLO biosynthesis disorders, and show that attenuation of polypeptide synthesis by PERK promotes extension of LLO intermediates to G(3)M(9)Gn(2)-P-P-Dol under these substrate-limiting conditions, as well as counteract abnormal N-glycosylation. This simple mechanism requires eIF2alpha Ser(51) phosphorylation by PERK, and is mimicked by agents that stimulate cytoplasmic stress-responsive Ser(51) kinase activity. Thus, by sensing ER stress from defective glycosylation, PERK can restore ER homeostasis by balancing polypeptide synthesis with flux through the LLO pathway

In response to alphabeta1 integrin signaling, transducers such as focal adhesion kinase (FAK) become activated, relaying to specific machineries and triggering distinct cellular responses. By conditionally ablating Fak in skin epidermis and culturing Fak-null keratinocytes, we show that FAK is dispensable for epidermal adhesion and basement membrane assembly, both of which require alphabeta1 integrins. FAK is also dispensible for proliferation/survival in enriched medium. In contrast, FAK functions downstream of alphabeta1 integrin in regulating cytoskeletal dynamics and orchestrating polarized keratinocyte migration out of epidermal explants. Fak-null keratinocytes display an aberrant actin cytoskeleton, which is tightly associated with robust, peripheral focal adhesions and microtubules. We find that without FAK, Src, p190RhoGAP, and PKL-PIX-PAK, localization and/or activation at focal adhesions are impaired, leading to elevated Rho activity, phosphorylation of myosin light chain kinase, and enhanced tensile stress fibers. We show that, together, these FAK-dependent activities are critical to control the turnover of focal adhesions, which is perturbed in the absence of FAK

A prevailing view regarding the regulation of connexin43 (Cx43) gap junction channels is that, upon intracellular acidification, the carboxyl-terminal domain (Cx43CT) moves toward the channel opening to interact with specific residues acting as a receptor site. Previous studies have demonstrated a direct, pH-dependent interaction between the Cx43CT and a Cx43 cytoplasmic loop (Cx43CL) peptide. This interaction was dependent on alpha-helical formation for the peptide in response to acidification; more recent studies have shown that acidification also induces Cx43CT dimerization. Whether Cx43CT dimerization is an important structural component in Cx43 regulation remains to be determined. Here we used an assortment of complimentary biophysical techniques to characterize the binding of Cx43CT or its mutants to itself and/or to a more native-like Cx43CL construct (Cx43CL(100-155), residues 100-155). Our studies expand the observation that specific Cx43CT domains are important for dimerization. We further show that properties of the Cx43CL(100-155) are different from those of the Cx43CL peptide; solvent acidification leads to Cx43CL(100-155) oligomerization and a change in the stoichiometry and binding affinity for the Cx43CT. Homo-Cx43CT and Cx43CL(100-155) oligomerization as well as the Cx43CT/Cx43CL(100-155) interaction can occur under in vivo conditions; moreover, we show that Cx43CL(100-155) strongly affects resonance peaks corresponding to Cx43CT residues Arg-376-Asp-379 and Asn-343-Lys-346. Overall, our data indicate that many of the sites involved in Cx43CT dimerization are also involved in the Cx43CT/Cx43CL interaction; we further propose that chemically induced Cx43CT and Cx43CL oligomerization is important for the interaction between these cytoplasmic domains, which leads to chemically induced gating of Cx43 channels

Recent single-molecule micromanipulation experiments on DNA subject to small distortion revealed positive coupling between DNA stretching and twisting--for instance, DNA elongates when overtwisted. Here we propose a method to calculate the twist-stretch coupling constant specific to a DNA fragment of a given sequence. The method employs a sequence-dependent dinucleotide force field and is based on constrained minimization of the fragment's deformation energy. Using a force field inferred from atomistic molecular dynamics simulations, we obtain the twist-stretch coupling for random sequence to be 0.30 nm/turn, close to experimental values. An exhaustive calculation for all oligomers of nine basepairs yields values between 0.14 and 0.45 nm/turn, positively correlated with the contents of pyrimidine-purine steps in the sequence. Our method is simple to use and allows one to explore the hypothesis that some sequences may be optimized for twist-stretch coupling.

HIV DNA vaccines are potent inducers of cell-mediated immune (CMI) response in mice but elicit poor HIV-specific IFN-gamma-producing T cells in monkeys and humans. In this study, we performed kinetic analyses on splenocytes of BALB/c mice that were immunized by a single injection with a unique DNA vaccine. Using IFN-gamma-ELISPOT and multiparametric FACS analysis, we characterized the induced CMI response. We found that the response was detectable for at least 63 wk. ELISPOT detection of IFN-gamma-producing T cells showed a profile with two waves separated by a long period of minimal response. Multiparametric FACS analysis showed two populations of CD3(+)CD8(+) T cells that were specific for all HIV Ags. These cells had similar robust proliferation abilities and contained granzyme B. However, only a few produced IFN-gamma. Both IFN-gamma-producing and non-IFN-gamma-producing HIV-specific CD8(+) T cells were detected in the early stage (week (W)1 and W2 postimmunization (PI)), in the prolonged intermediate period of minimal response (W4-W26 PI), and in the final late phase of increased response (W30-W63 PI). Our longitudinal characterization showed that both subsets of cells underwent expansion, contraction, and memory generation/maintenance phases throughout the lifespan of the animal. Altogether, these findings bring insight to the heterogeneity of the immune T cell response induced by a single immunization with this DNA and strengthen the concept that used of the IFN-gamma-ELISPOT assay alone may be insufficient to detect critical T cell responses to candidate HIV vaccines.

BACKGROUND: Fibroblast growth factor receptor 1 (FGFR1) mRNA can be alternatively spliced to generate isoforms containing (FGFR1alpha) or lacking (FGFR1beta) the first immunoglobulin-like domain. We examined which isoforms are expressed by cultured prostate cells, their affinities for FGF-2, and the effect of heparin on FGF-2 binding. METHODS: FGFR1 isoform expression was examined by RT-PCR. FGFR1alpha and FGFR1beta were expressed in CHO cells mutant in heparan sulfate synthesis, and their affinities for FGF-2, FGF-1, FGF-4, and FGF-6 were determined in the presence and absence of heparin. RESULTS: FGFR1alpha was expressed in luminal epithelial cells, whereas FGFR1beta was expressed in basal epithelial and smooth muscle cells. FGFR1beta bound FGF-2 with three-fourfold higher affinity than FGFR1alpha both in the presence and absence of heparin. Heparin increased affinity of both receptor isoforms for FGF-2 approximately four-fivefold. CONCLUSIONS: Prostate smooth muscle and basal epithelial cells are likely to be more sensitive than luminal epithelial cells to the low concentrations of FGFs present in vivo

We present a fast, versatile and adaptive-multiscale algorithm for analyzing a wide-variety of DNA microarray data. Its primary application is in normalization of array data as well as subsequent identification of 'enriched targets', e.g. differentially expressed genes in expression profiling arrays and enriched sites in ChIP-on-chip experimental data. We show how to accommodate the unique characteristics of ChIP-on-chip data, where the set of 'enriched targets' is large, asymmetric and whose proportion to the whole data varies locally. SUPPLEMENTARY INFORMATION: Supplementary figures, related preprint, free software as well as our raw DNA microarray data with PCR validations are available at http://www.math.umn.edu/~lerman/supp/bioinfo06 as well as Bioinformatics online

Little is known about the formation of the interventricular septum (IVS), a central event during cardiogenesis. Here, we describe a novel population of myocardial progenitor cells in the primitive ventricle of the mouse embryo, which is characterized by expression of lysozyme M (lysM). Using LysM-Cre mice we show that lysozyme expressing cells give rise to the IVS and to a part of the left ventricular free wall, demonstrating that these heart regions are developmentally related. LysM+ precursors are not of hematopoietic origin and develop in the absence of transcription factors that regulate lysozyme expression in macrophages. LysM-deficient mice lack an overt cardiac phenotype, perhaps due to compensation by the related lysozyme P, which we also found to be expressed in the developing heart. Direct visualization of lysM expression, using LysM-EGFP knock-in mice, showed that ventricular septation is initiated at embryonic day 9 by the movement of myocardial trabeculae from the primitive ventricle towards the bulbo-ventricular groove and revealed the dynamics of IVS formation at later stages. Our studies predict that LysM-Cre mice will be useful to inactivate genes in the developing IVS

Earlier studies suggested that TSP50 is a testis-specific gene that encodes a protein, which is homologous to serine proteases but differs in that threonine replaces serine in its catalytic triad. Most importantly, it was abnormally reactivated in many breast cancer biopsies tested. While further investigating its biochemical and cell biological natures, we found that TSP50 exhibited enzyme activity and was located in the endoplasmic reticulum and cytosol membrane. During our studies to elucidate the regulatory mechanisms related to its differential expression, we discovered a putative p53-binding site and several Sp1-binding sites in the TSP50 promoter, which led us to test if it was regulated by the p53 gene. We found that the p53 transgene negatively regulated the TSP50 promoter in diverse types of cell lines. This result was consistent with other observations: (a) p53 overexpression reduced endogenous TSP50 expression; and (b) breast cancer cell lines containing mutated p53, such as MCF7/Adr, or normal p53, such as MCF7, produced high or low levels of TSP50 transcripts, which was consistent with the fact that TSP50 promoter activity was much higher in MCF7/Adr than that in MCF7 cells. We also found that the quantity of Sp1 transcription factor was lower in MCF7/Adr than in MCF7 cells, which suggested that another mechanism (i.e., transcription factor modulation) was also involved in TSP50 differential expression.