Katarzyna Augoff

Human DHHC proteins: a spotlight on the hidden player of palmitoylation.

Palmitoylation is one of the most common posttranslational lipid modifications of proteins and we now know quite a lot about it. However, the state of knowledge about the enzymes that catalyze this process is clearly insufficient. This review is focused on 23 human DHHC genes and their products - protein palmitoyltransferases. Here we describe mainly the structure and function of these proteins, but also, to a lesser degree, what the substrates of the enzymes are and whether they are related to various diseases. The main aim of this review was to catalogue existing information concerning the human DHHC family of genes/proteins, making them and their functions easier to understand.

Membrane rafts as a novel target in cancer therapy.

Membrane rafts are distinct plasma membrane microdomains that are enriched in sphingolipids and cholesterol. They organize receptors and their downstream molecules and regulate a number of intracellular signaling pathways. This review presents information on the dependence of several growth factor receptor signaling pathways on membrane rafts. It also discusses the involvement of rafts in the regulation of differentiation, apoptosis and cell migration connected with invasiveness and metastasis. Examples of known synthetic and naturally occurring substances that are known to affect lateral membrane organization in tumor cell growth are discussed as potential or actual therapeutics.

Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells.

Background: Lateral membrane organization is important in many essential cellular functions. Results: Lack of palmitoylation of normal or unknown anemia erythrocyte membrane proteins, specifically MPP1, leads to changes in lateral membrane organization. Conclusion: Palmitoylation of MPP1 is crucial for membrane organization and is linked to the pathogenesis of hemolytic anemia. Significance: This might be the first mechanism of biological control of membrane lateral organization. S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.

Expression of Decorin in Esophageal Cancer in Relation to the Expression of Three Isoforms of Transforming Growth Factor-Beta (TGF-β1, -β2, and -β3) and Matrix Metalloproteinase-2 Activity

To determine the role of the reactive stroma in cancer progression, we investigated decorin (DCN) and transforming growth factor-beta (TGF-β expression, and matrix metalloproteinase-2 (MMP-2) activity in the tumorous esophagus. We found statistically insignificantly decreased levels of DCN expression in the pathological tissues. No obvious alterations in TGF-β expression were noticed. The highly significant increase in MMP-2 activity in cancers did not result in elevated levels of TGF-β dimers. Therefore, the system of TGF-β liberation from its complex with DCN by activated MMP-2 does not seem to contribute to esophageal cancerogenesis, although this hypothesis should be reevaluated with a larger study group.

The role of MPP1/p55 and its palmitoylation in resting state raft organization in HEL cells

Here we show the crucial role of MPP1 in lateral membrane ordering/organization in HEL cells (derived from erythroid precursors). Biochemical analyses showed that inhibition of MPP1 palmitoylation or silencing of the MPP1 gene led to a dramatic decrease in the DRM fraction. This was accompanied by a reduction of membrane order as shown by fluorescence-lifetime imaging microscopy (FLIM) analyses. Furthermore, MPP1 knockdown significantly affects the activation of MAP-kinase signaling via raft-dependent RTK (receptor tyrosine kinase) receptors, indicating the importance of MPP1 for lateral membrane organization. In conclusion, palmitoylation of MPP1 appears to be at least one of the mechanisms controlling lateral organization of the erythroid cell membrane. Thus, this study, together with our recent results on erythrocytes, reported elsewhere (Łach et al., J. Biol. Chem., 2012, 287, 18974-18984), points to a new role for MPP1 and presents a novel linkage between membrane raft organization and protein palmitoylation.

A novel regulatory function of CDKN1A/p21 in TNFα-induced matrix metalloproteinase 9-dependent migration and invasion of triple-negative breast cancer cells

Metastasis is the leading cause of mortality in patients with highly invasive cancers and, as such, is a major problem for medicine. It has been increasingly recognized that cancer-related inflammation plays an important role in promoting invasion and the metastatic process in which cell motility and upregulation of proteolytic enzymes are crucial events. TNFα is a proinflammatory cytokine known to stimulate synthesis of MMP9, a zinc- and calcium-dependent endopeptidase contributing to the regulation of ECM remodeling and cell signaling. However, the precise molecular mechanism of TNFα-induced MMP9 gene expression in cancers is still not fully understood. This study shows that TNFα-induced cell migration and invasion involve ERK1/2-dependent up-regulation of CDKN1A/p21 expression in highly aggressive breast cancer cells and that CDKN1A/p21 plays an important regulatory role in TNFα-induced MMP9 gene expression, indicating an unknown function of CDKN1A/p21 as a regulator of proteolytic activity in cancer cells.

Fibroblast activating protein-α expression in squamous cell carcinoma of the esophagus in primary and irradiated tumors: the use of archival FFPE material for molecular techniques

There are numerous reports suggesting that fibroblast activating protein-α (FAP-α) plays an important role in invasion of various tumor types. We studied the expression pattern of FAP-α in esophageal squamous cell carcinoma (ESCC) patients who had not been treated primarily and those who had received neoadjuvant radiochemotherapy. Our goal was to establish whether readily available tissue specimens fixed in formalin and stored in paraffin blocks for years might still be a source of FAP-α RNA for PCR analysis. The study included 20 patients divided into two groups, 10 patients in each group. We evaluated the expression of FAP- α by PCR techniques in fresh frozen and in paraffin-embedded tissues, and compared it to the expression in non-cancer tissues. To detect the protein expression level of FAP-α in paraffin-embedded tissues we used chromogenic immunohistochemical (IHC) staining. Data were analyzed by t-test or the nonparametric Wilcoxon matched pair test using Statistica 12.5 software. We observed an increased level of the FAP-α gene and protein expression in cancer tissues when compared with their corresponding normal tissues. However, statistically significant differences were found only in the group of patients untreated before surgery. RNA extracted from paraffin-embedded tissue sections had very low quality, especially in the context of degradation. FAP-α remains a highly altered participant of a complex microenvironment in esophageal squamous cell carcinoma, and its role in cell signaling requires further study. In this paper, we conclude that the use of a regular RT-PCR method for diagnostic purposes, which we have presented in an earlier paper, can be as good as qRT-PCR. Also, immunohistochemistry proved to be very useful and the only reliable method that can be used on longterm stored formalin-fixed, paraffin-embedded tissues.

Achalasia – balloon dilation or surgery?

Background The optimal therapeutic schedule in patients with achalasia is still under discussion. The aim of this study was to review our institution’s experience with myotomy and dilation in patients with achalasia. Material/Methods Clinical data were available for 59 patients who had ever had myotomy (n=38), dilation (n=21), or both procedures (n=8) between 2000 and 2007. Patients were followed prospectively with objective (a barium esophagogram) and subjective (a simple survey that scored dysphagia and overall patient satisfaction with the procedure) diagnostic tools. In the group of patients after pneumatic dilatations, frequency of interventions was higher (1, 2) than in the myotomy group (0, 2) at 2-year follow-up. Patients after myotomy with recurrence of dysphagia were treated with dilations. Results Mean time of dysphagia occurrence was similar in both groups (10 months). The statistically significant differences in treatment outcomes in both groups were in favor of myotomy during 2-year follow-up. Conclusions The data indicates that both methods of treatment might be useful in dysphagia control, but better results are obtained after myotomy. Repeat interventions are more frequent after endoscopic dilation. One method of treatment does not exclude the other. A short period of symptom relief after myotomy may suggest the myotomy was incomplete.