No abstract

Prostate Specific Antigen (PSA) is a prostate-specific serine protease enzyme that hydrolyzes gel-forming proteins (semenogelins) and changes the semen from gel-like to watery viscosity, a process called semen liquefaction. Highly viscous semen and abnormal liquefaction reduce sperm motility and contribute to infertility. Previously, we showed that non-specific serine protease inhibitor (AEBSF) prevented proteolytic degradation of semenogelin in mice. However, it is unclear whether similar effect could be recapitulated in fresh human ejaculates. Therefore, in this study we evaluated the effect of AEBSF on the degradation of semenogelin (SEMG1) and its subsequent impact on semen liquefaction and sperm motility in fresh semen ejaculates collected from healthy men. We found that AEBSF showed a dual contraceptive action where it effectively 1) prevented degradation of SEMG1 resulting in viscous semen and 2) decreased sperm motility in human semen samples. However, the impact of AEBSF on sperm motility and viability could be due to its inhibitory activity toward other serine proteases or simply due to its toxicity. Therefore, to determine whether inhibition of PSA activity alone could disrupt SEMG1 degradation and contribute to hyperviscous semen, a neutralizing PSA antibody was used. We found that PSA antibody effectively prevented SEMG1 degradation with a subtle impact on sperm motility. These findings suggest that the target inhibition of PSA activity can prevent proteolytic degradation of SEMG1 and block liquefaction process, resulting in hyperviscous semen. As it is currently unknown if blocking semen liquefaction alone could prevent pregnancy, it needs further extensive studies before drawing any translational conclusions.

Palmitoylation refers to the attachment of palmitate to protein cysteine residues and assists with protein localization to the plasma membrane. Like protein phosphorylation, palmitoylation is often a dynamic process, and it involves the attachment of the palmitate by palmitoyl transferases and removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Currently, the techniques to study palmitoylation are difficult to perform and involve the use of harsh chemical reagents like hydroxylamine (HAM) to depalmitate proteins, often leading to destruction of the proteins being studied. Recombinant, purified PPT/APTs could be used as an alternative reagent to depalmitate proteins and also to investigate their individual specificities in depalmitoylation reactions. To purify APTs and PPTs, the coding sequences of human variants were PCR amplified, cloned and expressed to produce either GST or MBP‐tagged fusion proteins. Expressed proteins have been analyzed using SDS‐PAGE and bands of expected molecular mass have been identified. Enzyme activity is currently being tested with the substrate 4‐methylumbelliferyl‐6‐thiopalmitoyl‐b‐glucoside (MU‐6S‐palm‐bGlc) in a fluorogenic enzyme assay, with the goal of eventually using the fusion proteins in depalmitolyation reactions involving palmitoylated protein substrates.Support or Funding InformationThis work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TCL is a poorly characterized Rho‐family GTPase; however, our lab has performed detailed structure/function analysis of TCL to gain insight into its cellular biochemistry. We have found that its unique N‐terminal extension promotes GTP‐loading of TCL and plasma membrane localization. Deletion of this TCL‐specific sequence promotes localization of TCL to intracellular vesicles. To further explore the role of the N‐terminal and C‐terminal sequences on TCL localization, plasmids encoding Venus were manipulated to include amino acids 1–24 of TCL at the N‐terminus of Venus and amino acids 198–214 at the C‐terminus of Venus. Additional N‐ and C‐terminus deletion mutants of YFP‐tagged TCL were also produced. These plasmids have been transfected into HeLa cells, and subcellular localization of the expressed proteins assessed by fluorescence microscopy and a fractionation procedure. Our results show a clear role for the C‐ terminus of TCL in facilitating membrane localization, but no significant localization directly related to the N‐terminus. Defining how TCL localizes to different cellular membrane environments will help us to understand more clearly its role focal adhesion dynamics, as well as how these dynamics may relate to melanoma development and tumor‐associated angiogenesis. Support or Funding Information This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).

Palmitoylation is a dynamic process for regulating protein function and localization. It involves the attachment of palmitate to protein cysteine residues by palmitoyl transferases and its removal by palmitoyl protein thioesterase (PPTs) or acyl protein thioesterases (APTs). Techniques to study palmitoylation are difficult to perform and use harsh depalmitoylating reagents like hydroxylamine (HAM), often leading to destruction of the protein being studied. Using recombinant or over‐expressed PPT/APTs could be used as an alternative to HAM for studying protein palmitoylation/depalmitoylation dynamics, as well as investigating PPT/APT substate specificities. We have cloned human APT and PPT cDNA for the production of GST or MBP‐tagged fusion proteins to be used in acyl‐biotin exchange assays. We have also produced mCherry‐tagged versions for co‐expression with a target protein of interest to evaluate their activities. In these experiments, we will focus on the Rho GTPase TCL/RhoJ, which was recently shown to associate with the plasma membrane through palmitoylation. Cherry‐tagged APT/PPTs will be co‐transfected with YFP‐tagged TCL to determine if TCL membrane association diminishes. Overall, the goal of these experiments is to improve techniques for studying protein palmitoylation/depalmitoylation. Support or Funding Information This work was supported by Bemidji State University Biology Department and the College of Business, Mathematics, and Science. Support was also provided through the Neilson Foundation, Bemidji, MN and Regenerative Medicine Minnesota (RMM‐2017‐EP‐04).