Biochemistry, Biophysics, and Structural Biology Commons^™

Hyaluronan Facilitates Invasion Of Colon Carcinoma Cells In Vitro Via Interaction With Cd44, Hyeong-Rok Kim, Marie A. Wheeler, Christopher M. Wilson, Joji Iida, David Eng, Melanie A. Simpson, James B. Mccarthy, Kelli M. Bullard

Department of Biochemistry: Faculty Publications

Hyaluronan (HA) and its biosynthetic enzymes, HA synthases (HAS1, 2, and 3) are thought to participate in cancer progression. We have shown previously that HA production and HAS3 expression are increased in metastatic colon carcinoma cells (SW620) when compared with cells isolated from a primary tumor (SW480). Because invasion of the extracellular matrix is a fundamental event in tumor growth and metastasis, we hypothesized that SW620 cells would show greater invasive capability than SW480 cells, that invasion is HA dependent, and that HA mediates invasion via interaction with a cell-surface receptor. Invasion into artificial basement membrane (Matrigel) was assessed in …

Melanoma Chondroitin Sulfate Proteoglycan Enhances Fak And Erk Activation By Distinct Mechanisms, Jianbo Yang, Matthew A. Price, Cheryl L. Neudauer, Christopher Wilson, Soldano Ferrone, Hong Xia, Joji Iida, Melanie A. Simpson, James B. Mccarthy

Department of Biochemistry: Faculty Publications

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal–regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes …

Characterization Of Human Udp-Glucose Dehydrogenase: Cys-276 Is Required For The Second Of Two Successive Oxidations, Brandi J. Sommer, Joseph J. Barycki, Melanie A. Simpson

Department of Biochemistry: Faculty Publications

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for K_m and V_max that agree well with those reported for a bovine homolog. We used crystal coordinates …

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Buan, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous class of ATP-independent chaperones believed to prevent irreversible protein aggregation and to facilitate subsequent protein renaturation in cooperation with ATP-dependent chaperones. Although sHSP chaperone activity has been studied extensively in vitro, understanding the mechanism of sHSP function requires identification of proteins that are sHSP substrates in vivo. We have used both immunoprecipitation and affinity chromatography to recover 42 proteins that specifically interact with Synechocystis Hsp16.6 in vivo during heat treatment. These proteins can all be released from Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones and are heat-labile. Thirteen …

Tissue-Specific Expression And Developmental Regulation Of Cytochrome B561 Genes In Arabidopsis Thaliana And Raphanus Sativus, Wim Verelst, Jyoti Kapila, Janice De Almeida Engler, Julie M. Stone, Roland Caubergs, Han Asard

Department of Biochemistry: Faculty Publications

Ascorbate (Asc) is an essential molecule in many aspects of development and stress responses in plants and animals. Cytochromes b561 (cyts b561) are tightly coupled to Asc homeostasis. These proteins are found in mammalian tissues, where they are involved in the regeneration of Asc, serving the synthesis of catecholamine neurotransmitters, and in intestinal iron reduction. Plant genomes encode homologous membrane-associated, Asc-reducible cyts b561. The expression of these proteins in plants, however, has so far not been studied. We have now examined the expression of two Arabidopsis thaliana cyt b561-encoding genes—Artb561-1 and Artb561-2—using relative-quantitative RT-PCR …

Endocytic Function, Glycosaminoglycan Specificity, And Antibody Sensitivity Of The Recombinant Human 190-Kda Hyaluronan Receptor For Endocytosis (Hare), Ed Harris, Janet A. Weigel, Paul H. Weigel

Department of Biochemistry: Faculty Publications

The human hyaluronan receptor for endocytosis (hHARE) mediates the endocytic clearance of hyaluronan (HA) and chondroitin sulfate from lymph fluid and blood. Two hHARE isoforms (190 and 315 kDa) are present in sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339–349). Here we report the specificity and function of the 190-kDa HARE, expressed without the larger isoform, in Flp-In 293 cell lines (190hHARE cells). Like the native protein, recombinant hHARE contains ~25 kDa of N-linked oligosaccharides, binds HA in a ligand blot …

5-Hydroxydecanoate Is Metabolised In Mitochondria And Creates A Rate-Limiting Bottleneck For Β-Oxidation Of Fatty Acids, Peter J. Hanley, Stefan Dröse, Ulrich Brandt, Rachel A. Lareau, Abir L. Banerjee, D. K. Srivastava, Leonard J. Banaszak, Joseph J. Barycki, Paul P. Van Veldhoven, Jürgen Daut

Department of Biochemistry: Faculty Publications

5-Hydroxydecanoate (5-HD) blocks pharmacological and ischaemic preconditioning, and

has been postulated to be a specific inhibitor of mitochondrial ATP-sensitive K+ (KATP)

channels. However, recent work has shown that 5-HD is activated to 5-hydroxydecanoyl-CoA

(5-HD-CoA), which is a substrate for the first step of β-oxidation. We have now

analysed the complete β-oxidation of 5-HD-CoA using specially synthesised (and purified)

substrates and enzymes, as well as isolated rat liver and heart mitochondria, and compared

it with the metabolism of the physiological substrate decanoyl-CoA. At the second step of

β-oxidation, catalysed by enoyl-CoA hydratase, enzyme kinetics were …

Interactions Between Small Heat Shock Protein Subunits And Substrate In Small Heat Shock Protein-Substrate Complexes, Kenneth L. Friedrich, Kim C. Giese, Nicole R. Baun, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

Small heat shock proteins (sHSPs) are dynamic oligomeric

proteins that bind unfolding proteins and protect

them from irreversible aggregation. This binding results

in the formation of sHSP-substrate complexes from

which substrate can later be refolded. Interactions between

sHSP and substrate in sHSP-substrate complexes

and the mechanism by which substrate is transferred to

ATP-dependent chaperones for refolding are poorly defined.

We have established C-terminal affinity-tagged

sHSPs from a eukaryote (pea HSP18.1) and a prokaryote

(Synechocystis HSP16.6) as tools to investigate these issues.

We demonstrate that sHSP subunit exchange for

HSP18.1 and HSP16.6 is temperature-dependent and

rapid at the optimal growth …

Interactions Between Small Heat Shock Protein Subunits And Substrate In Small Heat Shock Protein-Substrate Complexes, Kenneth L. Friedrich,, Kim C. Giese, Nicole R. Buan, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

Small heat shock proteins (sHSPs) are dynamic oligomeric proteins that bind unfolding proteins and protect them from irreversible aggregation. This binding results in the formation of sHSP-substrate complexes from which substrate can later be refolded. Interactions between sHSP and substrate in sHSP-substrate complexes and the mechanism by which substrate is transferred to ATP-dependent chaperones for refolding are poorly defined. We have established C-terminal affinity-tagged sHSPs from a eukaryote (pea HSP18.1) and a prokaryote (Synechocystis HSP16.6) as tools to investigate these issues. We demonstrate that sHSP subunit exchange for HSP18.1 and HSP16.6 is temperature-dependent and rapid at the optimal growth temperature …

Mobilization Of Intracellular Copper Stores By The Ctr2 Vacuolar Copper Transporter, Erin M. Rees, Jaekwon Lee, Dennis J. Thiele

Department of Biochemistry: Faculty Publications

Copper plays an essential role in processes including signaling to the transcription and protein trafficking machinery, oxidative phosphorylation, iron mobilization, neuropeptide maturation, and normal development. Whereas much is known about intracellular mobilization of ions such as calcium, little information is available on how eukaryotic cells mobilize intracellular copper stores. We describe a mechanism by which the Saccharomyces cerevisiae Ctr2 protein provides bioavailable copper via mobilization of intracellular copper stores. Whereas Ctr2 exhibits structural similarity to the Ctr1 plasma membrane copper importer, microscopic and biochemical fractionation studies localize Ctr2 to the vacuole membrane. We demonstrate that Ctr2 mobilizes vacuolar copper stores …

A Role For Talin In Presynaptic Function, Jennifer R. Morgan, Gilbert Di Paolo, Hauke Werner, Valentina A. Shchedrina, Marc Pypaert, Vincent A. Pieribone, Pietro De Camilli

Department of Biochemistry: Faculty Publications

Talin, an adaptor between integrin and the actin cytoskeleton at sites of cell adhesion, was recently found to be present at neuronal synapses, where its function remains unknown. Talin interacts with phosphatidylinositol-(4)-phosphate 5-kinase type Iγ, the major phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]–synthesizing enzyme in brain. To gain insight into the synaptic role of talin, we microinjected into the large lamprey axons reagents that compete the talin–PIP kinase interaction and then examined their effects on synaptic structure. A dramatic decrease of synaptic actin and an impairment of clathrin-mediated synaptic vesicle endocytosis were observed. The endocytic defect included an accumulation of clathrin-coated pits …

Methionine Sulfoxide Reductase Regulation Of Yeast Lifespan Reveals Reactive Oxygen Species-Dependent And -Independent Components Of Aging, Ahmet Koc, Audrey P. Gasch, Julian C. Rutherford, Hwa-Young Kim, Vadim N. Gladyshev

Department of Biochemistry: Faculty Publications

Aging is thought to be caused by the accumulation of damage, primarily from oxidative modifications of cellular components by reactive oxygen species (ROS). Here we used yeast methionine sulfoxide reductases MsrA and MsrB to address this hypothesis. In the presence of oxygen, these antioxidants could increase yeast lifespan and did so independent of the lifespan extension offered by caloric restriction. However, under ROS-deficient, strictly anaerobic conditions, yeast lifespan was shorter, not affected by MsrA or MsrB, and further reduced by caloric restriction. In addition, we identified changes in the global gene expression associated with aging in yeast, and they did …

The Identity Of Proteins Associated With A Small Heat Shock Protein During Heat Stress In Vivo Indicates That These Chaperones Protect A Wide Range Of Cellular Functions, Eman Basha, Garrett J. Lee, Linda A. Breci, Andrew C. Hausrath, Nicole R. Baun, Kim C. Giese, Elizabeth Vierling

Department of Biochemistry: Faculty Publications

The small heat shock proteins (sHSPs) are a ubiquitous

class of ATP-independent chaperones believed to

prevent irreversible protein aggregation and to facilitate

subsequent protein renaturation in cooperation

with ATP-dependent chaperones. Although sHSP chaperone

activity has been studied extensively in vitro, understanding

the mechanism of sHSP function requires

identification of proteins that are sHSP substrates in

vivo. We have used both immunoprecipitation and affinity

chromatography to recover 42 proteins that specifically

interact with Synechocystis Hsp16.6 in vivo during

heat treatment. These proteins can all be released from

Hsp16.6 by the ATP-dependent activity of DnaK and cochaperones

and are heat-labile. …