Development and Validation of an Enzymatic Assay for TMPRSS4: Evaluation of Molecular Inhibitors

TMPRSS4, a member of the transmembrane serine protease (TTSP) family, has earned significant attention due to its pronounced overexpression in various carcinoma types, involvement in disease processes, and viral entry.  However, despite its pivotal role in disease biology, little is known about its structural characteristics and potential drug targets. Accurate measurement of TMPRSS4 activity is crucial for understanding its role in these disease processes and for developing potential therapeutic interventions. In this context, we have expressed and purified TMPRSS4 protein and report the development of an enzymatic assay for the quantitative measurement of TMPRSS4 activity.  The assay is based on a synthetic fluorogenic peptide substrate that mimics the natural cleavage site of TMPRSS4/serine proteases substrates. The Assay development workflow includes enzyme preparation, optimization of assay materials, reagents, and conditions.  The assay is further validated using a known serine protease inhibitor, Aprotinin. Our results demonstrate that the developed enzymatic assay exhibits a wide dynamic range and successfully applied the assay to assess molecular inhibitors predicted by our computational studies. In conclusion, our study presents a robust enzymatic assay for TMPRSS4 activity that provides researchers and clinicians with a valuable tool for studying this protease's function and potential clinical applications

Comprehensive Investigation of Recombinant Human TMPRSS4 Expression and Purification Across Diverse Expression Platforms: Bacterial, Insect (BVES), and Mammalian Systems

TMPRSS4, an essential transmembrane protease serine 4, holds significant relevance in diverse biological contexts, making it a molecule of interest across various fields. This transmembrane serine protease, TMPRSS4, plays a pivotal role in multiple areas, including cancer research, disease processes, and potentially in the spread of viral infections.  In light of its multifaceted significance, the research and production of active TMPRSS4 protein serve as a critical foundation for in-depth structural and functional studies, ultimately leading to the identification of potential inhibitors. Within this context, our study encompasses an extensive investigation of various expression systems, including microbial, insect cells, and mammalian cells, aimed at obtaining biologically active TMPRSS4 protein. This study underscores our efforts in optimizing solubility. We explored different E. coli expression strains, optimized expression conditions at various temperatures, employed both inducible and autoinduction-based approaches, fine-tuned the Multiplicity of Infection (MOI), and controlled cell density in Sf9 cells using BVES. Additionally, we delved into the exploration of alternative signal peptides for secretory expression in mammalian cells, followed by the characterization of purified protein variants. Through rigorous biochemical analyses and functional assays, we discovered the distinct advantages associated with mammalian cell expression, ultimately resulting in the production of biologically active TMPRSS4. Our findings provide critical insights into the optimization of expression systems, thereby enhancing the functionality of the protein

Analysis of LTE Radio Frame by eliminating Cyclic Prefix in OFDM and comparison of QAM and Offset-QAM

Spectral efficiency is the key factor for the development of future wireless communications. Orthogonal Frequency Division Multiple Access (OFDMA) is the multiple access technology used at physical layer of latest wireless communication technologies. Anything on the improvement or overcoming the disadvantage of the present system will be considered for the future wireless systems. Long Term Evolution (LTE) is one of the 4th generation wireless communications and it is taken as the reference system in this thesis. The main concern of this thesis is to analyze the LTE radio frame. We designed and simulated the OFDM system with cyclic prefix, its Bit Error Rate (BER) is verified by changing the Signal to Noise Ratio (SNR) value and we investigated the OFDM system by eliminating the cyclic prefix. By eliminating cyclic prefix bandwidth efficiency is achieved, though using cyclic prefix in OFDM has more advantages. Filter banks are used to compensate the advantages of cyclic prefix when it is removed. Introducing Offset in QAM results in less distortion and amplitude fluctuations. We designed, simulated and compared the QAM digital modulation with Offset-QAM digital modulation its BER vs. SNR are verified using simulations on MATLAB

HIV-1 Nef-induced FasL induction and bystander killing requires p38 MAPK activation

The human immunodeficiency virus (HIV) has been reported to target noninfected CD4 and CD8 cells for destruction. This effect is manifested in part through up-regulation of the death receptor Fas ligand (FasL) by HIV-1 negative factor (Nef), leading to bystander damage. However, the signal transduction and transcriptional regulation of this process remains elusive. Here, we provide evidence that p38 mitogen-activated protein kinase (MAPK) is required for this process. Loss-of-function experiments through dominant-negative p38 isoform, p38 siRNA, and chemical inhibitors of p38 activation suggest that p38 is necessary for Nef-induced activator protein-1 (AP-1) activation, as inhibition leads to an attenuation of AP-1-dependent transcription. Furthermore, mutagenesis of the FasL promoter reveals that its AP-1 enhancer element is required for Nef-mediated transcriptional activation. Therefore, a linear pathway for Nef-induced FasL expression that encompasses p38 and AP-1 has been elucidated. Furthermore, chemical inhibition of the p38 pathway attenuates HIV-1-mediated bystander killing of CD8 cells in vitro. (Blood. 2005;106:2059-2068

HIV-1 viral protein-R (VPR) protects against lethal superantigen challenge while maintaining homeostatic T cell levels in vivo

The HIV-1 accessory protein Vpr exhibits many interesting features related to macrophage and T cell biology. As a viral protein or as a soluble molecule it can suppress immune cell activation and cytokine production in vitro in part by targeted inhibition of NF-kappa B. In this regard we sought to test its effects in vivo on an NF-kappa B-dependent immune pathway. We examined the activity of Vpr in a lethal toxin-mediated challenge model in mice. Intravenous delivery of Vpr was sufficient to protect mice from lethal challenge with staphylococcal endotoxin B (SEB). Furthermore, Vpr protected host CD4(+) T cells from in vivo depletion likely by preventing induction of AICD of SEB-exposed cells in a post-toxin-binding fashion. Understanding the biology of Vpr's activities in this model may allow for new insight into potential mechanisms of hyperinflammatory disease and into Vpr pathobiology in the context of HIV infection