14 research outputs found
Glomerular expression profiling and novel proteins in normal mouse kidney and adriamycin-induced nephrosis
Kidney glomeruli function as high-capacity molecular sieves through which
plasma is filtered into the Bowman s space as primary urine. The
glomerular filtration barrier is composed of glomerular endothelial
cells, the glomerulus basement membrane and the podocyte cell layer.
Dysfunction of the glomerulus is a central component of renal
complications leading to end-stage renal disease. However, the molecular
composition of the glomerulus and how it changes during disease are still
mostly unknown. To elucidate the picture of molecules involved in the
biology and pathology of the glomerulus, large-scale approaches including
two-dimensional gel electrophoresis (2-DE) coupled with mass spectrometry
analysis and microarray profiling were applied to normal and diseased
glomeruli.
A proteome analysis of healthy glomeruli in mouse was performed using
2-DE with two different staining methods and subsequent mass
spectrometric identifications. Altogether, 414 protein spots were
identified, revealing 232 different proteins representing a wide spectrum
of activities. Only 53 of the proteins identified here were detected in
another proteome study, showing the value of analysis utilizing different
methodologies. 80 of the proteins were not identified in a separate
transcriptome analysis, while ten of the present proteins were identified
as genes implicated in glomerulus development and function, allowing
direct correlation with expression data.
Characterization of five glomerulus-upregulated transcripts/proteins,
ehd3, dendrin, sh2d4a, plekhh2, and 2310066E14Rik was performed. The
expression pattern of these novel glomerular transcripts in various mouse
tissues was studied, and the distribution of corresponding proteins was
examined. All five transcripts/proteins were expressed in the kidney
exclusively by glomerular cells. Ehd3 was expressed only by glomerular
endothelial cells. Importantly, ehd3 is the first gene ever shown to be
expressed exclusively by glomerular endothelial cells and not by other
endothelial cells in the kidney. Dendrin, sh2d4a, plekhh2, and
2310066E14Rik were transcribed specifically in podocytes within the
glomerulus. With the use of polyclonal antibodies, dendrin, sh2d4a, and
plekhh2 proteins were localized to the slit diaphragm and the foot
process, whereas 2310066E14Rik protein was localized to the podocyte
major processes and cell body.
Comparison of the normal glomerular transcriptome with its changes during
progression of glomerular disease can yield information about molecular
pathomechanisms. The adriamycin (ADR)-induced proteinuric mouse model
allows the precise timing of the onset of proteinuria and of
morphological changes in glomerulus. Overt proteinuria was observed from
four days after ADR injection, and reached maximum at seven days. Blood
urea nitrogen (BUN) gradually elevated indicating the failure of renal
function. TUNEL staining of kidney section revealed an increase in
apoptotic positive cells in glomeruli. Transcriptional profiling of
kidney glomeruli revealed that nine p53 target genes were up-regulated
probably due to DNA damage caused by ADR at four days and several
glomerular enriched genes were differentially expressed indicating
glomerular injury at seven days.
These studies provide fresh insights into glomerular biology and reveal
new possibilities to explore the role of glomerular specific proteins in
renal physiology and pathophysiology. Furthermore, these studies shed
light on the pathomechanisms of proteinuria, which eventually results in
end-stage kidney disease as a result of progressive glomerular damage
The Natural Flavonoid Compound Deguelin Inhibits HCMV Lytic Replication within Fibroblasts
Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus for which there is no vaccine or cure. This viral infection, once acquired, is life-long, residing latently in hematopoietic cells. However, latently infected individuals with weakened immune systems often undergo HCMV reactivation, which can cause serious complications in immunosuppressed and immunocompromised patients. Current anti-viral therapies target late stages of viral replication, and are often met with therapeutic resistance, necessitating the development of novel therapeutics. In this current study, we identified a naturally-occurring flavonoid compound, deguelin, which inhibits HCMV lytic replication. Our findings reveal that nanomolar concentrations of deguelin significantly suppress the production of the infectious virus. Further, we show that deguelin inhibits the lytic cycle during the phase of the replication cycle consistent with early (E) gene and protein expression. Importantly, our data reveal that deguelin inhibits replication of a ganciclovir-resistant strain of HCMV. Together, our findings identify a novel, naturally occurring compound that may prove useful in the treatment of HCMV replication
L-ascorbic acid-6-hexadecanoate, a potent hyaluronidase inhibitor: X-ray structure and molecular modeling of enzyme-inhibitor complexes.
Hyaluronidases are enzymes that degrade hyaluronan, an important component of the extracellular matrix. The mammalian hyaluronidases are considered to be involved in many (patho)physiological processes like fertilization, tumor growth, and metastasis. Bacterial hyaluronidases, also termed hyaluronate lyases, contribute to the spreading of microorganisms in tissues. Such roles for hyaluronidases suggest that inhibitors could be useful pharmacological tools. Potent and selective inhibitors are not known to date, although L-ascorbic acid has been reported to be a weak inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL). The x-ray structure of SpnHL complexed with L-ascorbic acid has been elucidated suggesting that additional hydrophobic interactions might increase inhibitory activity. Here we show that L-ascorbic acid 6-hexadecanoate (Vcpal) is a potent inhibitor of both streptococcal and bovine testicular hyaluronidase (BTH). Vcpal showed strong inhibition of Streptococcus agalactiae hyaluronate lyase with an IC(50) of 4 microM and weaker inhibition of SpnHL and BTH with IC(50) values of 100 and 56 microM, respectively. To date, Vcpal has proved to be one of the most potent inhibitors of hyaluronidase. We also determined the x-ray structure of the SpnHL-Vcpal complex and confirmed the hypothesis that additional hydrophobic interactions with Phe-343, His-399, and Thr-400 in the active site led to increased inhibition. A homology structural model of BTH was also generated to suggest binding modes of Vcpal to this hyaluronidase. The long alkyl chain seemed to interact with an extended, hydrophobic channel formed by mostly conserved amino acids Ala-84, Leu-91, Tyr-93, Tyr-220, and Leu-344 in BTH
Rhophilin-1 Is a Key Regulator of the Podocyte Cytoskeleton and Is Essential for Glomerular Filtration
Association of crumbs homolog-2 with mTORC1 in developing podocyte.
The evidence that gene mutations in the polarity determinant Crumbs homologs-2 (CRB2) cause congenital nephrotic syndrome suggests the functional importance of this gene product in podocyte development. Because another isoform, CRB3, was reported to repress the mechanistic/mammalian target of the rapamycin complex 1 (mTORC1) pathway, we examined the role of CRB2 function in developing podocytes in relation to mTORC1. In HEK-293 and MDCK cells constitutively expressing CRB2, we found that the protein localized to the apicolateral side of the cell plasma membrane and that this plasma membrane assembly required N-glycosylation. Confocal microscopy of the neonate mouse kidney revealed that both the tyrosine-phosphorylated form and non-phosphorylated form of CRB2 commence at the S-shaped body stage at the apicolateral side of podocyte precursor cells and move to foot processes in a capillary tuft pattern. The pattern of phosphorylated mTOR in developing podocytes was similar to that of CRB2 tyrosine phosphorylation. Additionally, the lack of a tyrosine phosphorylation site on CRB2 led to the reduced sensitivity of mTORC1 activation in response to energy starvation. CRB2 may play an important role in the mechanistic pathway of developing podocytes through tyrosine phosphorylation by associating with mTORC1 activation
Design of new benzoxazole-2-thione derived inhibitors of Streptococcus pneumoniae hyaluronan lyase: structure of a complex with a 2-phenylindole.
The bacterial hyaluronan lyases (Hyals) that degrade hyaluronan, an important component of the extracellular matrix, are involved in microbial spread. Inhibitors of these enzymes are essential in investigation of the role of hyaluronan and Hyal in bacterial infections and constitute a new class of antibiotics against Hyal-producing bacteria. Recently, we identified 1,3-diacetylbenzimidazole-2-thione and related molecules as inhibitors of streptococcal Hyal. One of such compounds, 1-decyl-2-(4-sulfamoyloxyphenyl)-1-indol-6-yl sulfamate, was co-crystallized in a complex with Streptococcus pneumoniae Hyal and its structure elucidated. The resultant X-ray structure demonstrates that this inhibitor fits in the enzymatic active site via interactions resembling the binding mode of the natural hyaluronan substrate. X-ray structural analysis also indicates binding interactions with the catalytic residues and those of a catalytically essential hydrophobic patch. An IC50 value of 11 microM for Hyal from Streptococcus agalactiae (strain 4755) qualifies this phenylindole compound as one of the most potent Hyal inhibitors known to date. The structural data suggested a similar binding mode for N-(3-phenylpropionyl)-benzoxazole-2-thione. This new compound's inhibitory properties were confirmed resulting in discovery of yet another Hyal inhibitor (IC50 of 15 microM). These benzoxazole-2-thiones constitute a new class of inhibitors of bacterial Hyals and are well suited for further optimization of their selectivity, potency, and pharmacokinetic properties
Expression and Subcellular Distribution of Novel Glomerulus-Associated Proteins Dendrin, Ehd3, Sh2d4a, Plekhh2, and 2310066E14Rik
Cytomegalovirus US28 regulates cellular EphA2 to maintain viral latency.
Cytomegalovirus (CMV) reactivation from latency following immune dysregulation remains a serious risk for patients, often causing substantial morbidity and mortality. Here, we demonstrate the CMV-encoded G protein-coupled receptor, US28, in coordination with cellular Ephrin receptor A2, attenuates mitogen-activated protein kinase signaling, thereby limiting viral replication in latently infected primary monocytes. Furthermore, treatment of latently infected primary monocytes with dasatinib, a Food and Drug Association-approved kinase inhibitor used to treat a subset of leukemias, results in CMV reactivation. These ex vivo data correlate with our retrospective analyses of the Explorys electronic health record database, where we find dasatinib treatment is associated with a significant risk of CMV-associated disease (odds ratio 1.58, P = 0.0004). Collectively, our findings elucidate a signaling pathway that plays a central role in the balance between CMV latency and reactivation and identifies a common therapeutic cancer treatment that elevates the risk of CMV-associated disease