Progestin functions in vertebrate gametes mediated by membrane progestin receptors (mPRs): Identification of mPRα on human sperm and its association with sperm motility

Most of the studies on the putative membrane progestin receptor (mPR) α and β subtypes that have been published in the 5 years since their discovery have supported the original hypothesis that they function as specific membrane receptors through which progestins induce rapid, nongenomic responses in target cells. Recent evidence that mPRα and mPRβ have important roles in the regulation of oocyte meiotic maturation and sperm motility in both fish and mammals is reviewed. Although rapid, cell surface-initiated progestin actions on sperm to induce hyperactive motility have been demonstrated in several mammalian models, the identity of the membrane progestin receptor mediating this effect remains unclear. We demonstrate here that mPRα mRNA is expressed in human sperm by RT-PCR and that the mPRα protein is localized to the sperm membranes by Western blot analysis. Immunocytochemical staining of whole non-permeabilized human sperm confirmed the mPRα protein is expressed in the plasma membrane, and showed it is localized to the sperm midpiece, indicating a likely role of mPRα in progestin regulation of sperm motility. Moreover, the abundance of the mPRα protein on sperm plasma membranes from human donors that displayed low motility was significantly reduced compared to that on normal motile sperm. Finally, progestin treatment of sperm membranes caused activation of G-proteins. These results suggest that, similar to its proposed function in fishes, mPRα is an intermediary in progestin stimulation of sperm motility in humans by a mechanism involving G-protein activation.     

A high-level expression vector containing selectable marker for continuous production of recombinant protein in insect cells

Insect cell expression systems are widely used to produce active recombinant proteins. Here, we have developed a high-level expression vector containing a selectable marker for continuous production of recombinant proteins in insect cells. The plasmid, pXIHAbla, developed in this study, established a polyclonal cell line 8 days shorter than pXINSECT-DEST38 and pBmAneo. In addition, pXIHAbla exhibited an approximately fivefold higher average enhanced GFP expression level and approximately a twofold higher bionanocapsule secretion level than pXINSECT-DEST38. Using this plasmid, insect cells that highly express active proteins have been easily established.     

Analysis of changes induced by oncogene <Emphasis Type="Italic">N-RAS</Emphasis> expression in pattern and distribution of pseudopodial activity of fibroblasts

It is not known which morphological properties of fibroblasts induced by malignant transformation modulate their migration pattern. We studied the changes in the distribution and dynamics of the leading edge of 10(3) mouse fibroblasts after their transformation by oncogene N-RAS ^asp13 and analyzed the changes in the pattern of cell migration. Transformation proved to increase the leading edge proportion and to considerably redistribute pseudopodial activity along the cell edge. As the result of transformation, small pseudopodia are formed in the stable lateral regions of the cell edge typical of normal fibroblasts, i.e., the lateral edge is no more truly stable. In addition, pseudopodial activity of the leading edge in transformed fibroblasts proved higher compared to normal ones. It is necessary to notice, the leading edge activity is equally high immediately after induction in both normal and transformed fibroblasts; although, it is suppressed with time in normal cells but not in transformed ones where it remains steadily high. These properties promote the random component of malignant cell motility and modify the cell migration pattern after transformation     

Expression of human interferon alpha-1 with enhanced stability via the tagging system of a stabilizing peptide

Human interferon α-1 (hIFNA1) is one of several interferon α subtypes that have been studied and commercialized to treat various viral diseases including hepatitis B and C as well as malignant melanoma. Protein aggregation has been problematic for every step in commercial production, from purification to the packaging and delivery of pharmaceutical proteins. In a previous study, we demonstrated that a stabilizing peptide from the C-terminal acidic tail of α-synuclein (ATS) could be used as an effective fusion tag to increase the stability of target proteins such as human growth hormone (hGH) and granulocyte colony-stimulating factor (G-CSF). In this study, we applied this ATS fusion system to hIFNA1 in order to protect against the aggregation of hIFNA1 by environmental stresses, since hIFNA1 aggregates elicit an undesirable immune response in humans. As expected, ATS-fused hIFNA1 (hIFNA1-ATS) protein showed enhanced stability against thermal stress, agitation stress, and repetitive freeze/thawing stress in comparison with native hIFNA1. More importantly, hIFNA1-ATS fusion protein appeared to be 1.6 times more active than hIFNA1 in a cell anti-proliferation assay. Furthermore, the solubility of hIFNA1-ATS appeared to be 1.7 times higher than that of native protein. Our results suggest that the ATS-tag system could be a useful means for protecting hIFNA1 protein from aggregation by various external stresses and could be used to increase the solubility of protein.     

Regulation of Actin Dynamics in Pollen Tubes: Control of Actin Polymer Level

Actin cytoskeleton undergoes rapid reorganization in response to internal and external cues. How the dynamics of actin cytoskeleton are regulated, and how its dynamics relate to its function are fundamental questions in plant cell biology. The pollen tube is a well characterized actin-based cell morphogenesis in plants. One of the striking features of actin cytoskeleton characterized in the pollen tube is its surprisingly low level of actin polymer. This special phenomenon might relate to the function of actin cytoskeleton in pollen tubes. Understanding the molecular mechanism underlying this special phenomenon requires careful analysis of actin-binding proteins that modulate actin dynamics directly. Recent biochemical and biophysical analyses of several highly conserved plant actin-binding proteins reveal unusual and unexpected properties, which emphasizes the importance of carefully analyzing their action mechanism and cellular activity. In this review, we highlight an actin monomer sequestering protein, a barbed end capping protein and an F-actin severing and dynamizing protein in plant. We propose that these proteins function in harmony to regulate actin dynamics and maintain the low level of actin polymer in pollen tubes.     

Negative regulation of the actin-regulating kinase Prk1p by patch localization-induced autophosphorylation

The Prk1 family of protein kinases are important regulators of endocytosis and actin cytoskeleton in some eukaryotic cells. In budding yeast, Prk1p phosphorylates numerous endocytic proteins including Pan1p and Sla1p. Prk1p has been observed to undergo autophosphorylation in vivo . In this study, we determined the sites and underlying role of the autophosphorylation. Two sites located in the noncatalytic region were identified to be the autophosphorylation sites. When the sites were mutated, the non-autophosphorylatable Prk1p phosphorylated Pan1p and Sla1p more efficiently than the wild-type kinase, suggesting a negative effect of the autophosphorylation. In addition, the dynamic properties of actin and the coat complex were also altered in the autophosphorylation mutant cells. Interestingly, the autophosphorylation of Prk1p was dependent on cortical localization of the kinase and could be induced by phosphorylated Sla1p. These results suggest that the autophosphorylation of Prk1p may represent a feedback mechanism possibly involved in fine-tuning the pace of progression during actin-coupled endocytosis.     

The carboxy‐terminal tail of connexin43 gap junction protein is sufficient to mediate cytoskeleton changes in human glioma cells

Connexin43 (Cx43) is a ubiquitously expressed member of the gap junction protein family that mediates gap junction intercellular communication (GJIC) by allowing exchange of cytosolic materials. Previous studies have used Cx43 truncated at the cytoplasmic tail (C‐tail) to demonstrate that the C‐tail is essential to regulate cell growth and motility. Therefore, the aim of our study was to delineate the respective role of the truncated Cx43 and the C‐tail in mediating Cx43‐dependent signaling. A truncated Cx43 expressing the channel part of the protein (TrCx43, amino acid 1–242) and a construct encompassing only the C‐tail from amino acid 243 (243Cx43) were transduced into LN18 human glioma cells. Our results showed that the ability of Cx43 to suppress growth was independent of GJIC as assessed by dye transfer, but was dependent on the presence of a rigid extracellular matrix. We further demonstrated that the C‐tail alone is sufficient to promote motility. Surprisingly, Cx43 is also able to increase migration in the absence of the C‐tail, suggesting the presence of at least two distinct signaling mechanisms utilized by Cx43 to affect motility. Finally, we used time‐lapse imaging to examine the behavior of migrating cells and it was apparent that the C‐tail was associated with a lamellipodia‐based migration not observed in either mock or TrCx43 expressing LN18 cells. Our study shows for the first time that a free C‐tail is sufficient to induce Cx43‐dependent changes in cell morphology and that Cx43 signaling is linked to the regulation of the actin cytoskeleton. J. Cell. Biochem. 110: 589–597, 2010. © 2010 Wiley‐Liss, Inc.     

Multiscale modeling of structural dynamics underlying force generation and product release in actomyosin complex

To decrypt the mechanistic basis of myosin motor function, it is essential to probe the conformational changes in actomyosin with high spatial and temporal resolutions. In a computational effort to meet this challenge, we have performed a multiscale modeling of the allosteric couplings and transition pathway of actomyosin complex by combining coarse‐grained modeling of the entire complex with all‐atom molecular dynamics simulations of the active site. Our modeling of allosteric couplings at the pre‐powerstroke state has pinpointed key actin‐activated couplings to distant myosin parts which are critical to force generation and the sequential release of phosphate and ADP. At the post‐powerstroke state, we have identified isoform‐dependent couplings which underlie the reciprocal coupling between actin binding and nucleotide binding in fast Myosin II, and load‐dependent ADP release in Myosin V. Our modeling of transition pathway during powerstroke has outlined a clear sequence of structural events triggered by actin binding, which lead to subsequent force generation, twisting of central β‐sheet, and the sequential release of phosphate and ADP. Finally we have performed atomistic simulations of active‐site dynamics based on an on‐path “transition‐state” myosin conformation, which has revealed significantly weakened coordination of phosphate by Switch II, and a disrupted key salt bridge between Switch I and II. Meanwhile, the coordination of MgADP by Switch I and P loop is less perturbed. As a result, the phosphate can be released prior to MgADP. This study has shed new lights on the controversy over the structural mechanism of actin‐activated phosphate release and force generation in myosin motor. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Hepcidin expression by human monocytes in response to adhesion and pro-inflammatory cytokines

Background

A previous urine proteomic analysis from our laboratory suggested that hepcidin may be a biomarker for lupus nephritis flare. Immunohistochemical staining of kidney biopsies from lupus patients showed that hepcidin was expressed by infiltrating renal leukocytes. Here we investigated whether inflammatory cytokines relevant to the pathogenesis of lupus nephritis and other glomerular diseases regulate hepcidin expression by human monocytes.

Methods

Human CD14+ monocytes were incubated with interferon alpha (IFNα), interferon gamma (IFNγ), interleukin-6 (IL6), interleukin-1 beta (IL1β), monocyte chemotactic factor-1 (MCP1), or tumor necrosis factor alpha (TNFα). Hepcidin expression was examined by real-time PCR and enzyme immunoassay.

Results

Monocyte hepcidin mRNA increased during adherence to the tissue culture wells, reaching a level 150-fold higher than baseline within 12 h of plating. After accounting for the effects of adhesion, monocytes showed time and dose-dependent up-regulation of hepcidin mRNA upon treatment with IFNα or IL6. One hour of incubation with IFNα or IL6 increased hepcidin mRNA 20 and 80-fold, respectively; by 24 h the mRNA remained 5- and 2.4-fold higher than baseline. IL1β, IFNγ, and MCP-1 did not affect monocyte hepcidin expression. TNFα inhibited hepcidin induction by IL6 in monocytes by 44%. After 24 h of treatment with IFNα or IL6, immunoreactive hepcidin production by monocytes increased 3- and 2.6-fold, respectively.

Conclusion

Human monocytes produce hepcidin in response to adhesion and the pro-inflammatory cytokines IFNα and IL6.

General significance

The appearance of hepcidin in the kidneys or urine during glomerular diseases may be from infiltrating monocytes induced to express hepcidin by adherence and exposure to pro-inflammatory cytokines found in the renal milieu.