NatF Contributes to an Evolutionary Shift in Protein N-Terminal Acetylation and Is Important for Normal Chromosome Segregation

N-terminal acetylation (N-Ac) is a highly abundant eukaryotic protein modification. Proteomics revealed a significant increase in the occurrence of N-Ac from lower to higher eukaryotes, but evidence explaining the underlying molecular mechanism(s) is currently lacking. We first analysed protein N-termini and their acetylation degrees, suggesting that evolution of substrates is not a major cause for the evolutionary shift in N-Ac. Further, we investigated the presence of putative N-terminal acetyltransferases (NATs) in higher eukaryotes. The purified recombinant human and Drosophila homologues of a novel NAT candidate was subjected to in vitro peptide library acetylation assays. This provided evidence for its NAT activity targeting Met-Lys- and other Met-starting protein N-termini, and the enzyme was termed Naa60p and its activity NatF. Its in vivo activity was investigated by ectopically expressing human Naa60p in yeast followed by N-terminal COFRADIC analyses. hNaa60p acetylated distinct Met-starting yeast protein N-termini and increased general acetylation levels, thereby altering yeast in vivo acetylation patterns towards those of higher eukaryotes. Further, its activity in human cells was verified by overexpression and knockdown of hNAA60 followed by N-terminal COFRADIC. NatF's cellular impact was demonstrated in Drosophila cells where NAA60 knockdown induced chromosomal segregation defects. In summary, our study revealed a novel major protein modifier contributing to the evolution of N-Ac, redundancy among NATs, and an essential regulator of normal chromosome segregation. With the characterization of NatF, the co-translational N-Ac machinery appears complete since all the major substrate groups in eukaryotes are accounted for

Prolongation of rat-to-mouse islets xenograft survival by co-transplantation of autologous IL-10 differentiated murine tolerogenic dendritic cells

Tolerogenic dendritic cells (DCs) represent a promising approach to promote transplantation tolerance. In this study, the potential of autologous bone marrow (BM)-derived murine DC to protect rat-to-mouse islets xenografts was analyzed

Microencapsulation of hepatocytes and mesenchymal stem cells for therapeutic applications

Encapsulated hepatocyte transplantation and encapsulated mesenchymal stem cell transplantation are newly developed potential treatments for acute and chronic liver diseases, respectively. Cells are microencapsulated in biocompatible semi-permeable alginate-based hydrogels. Microspheres protect cells against antibodies and immune cells, while allowing nutrients, small/medium size proteins and drugs to diffuse inside and outside the polymer matrix. Microencapsulated cells are assessed in vitro and designed for experimental transplantation and for future clinical applications. Here, we describe the protocol for microencapsulation of hepatocytes and mesenchymal stem cells within hybrid poly(ethylene glycol)-alginate hydrogels

Design And Application Of Squeeze Film Dampers For Turbomachinery Stabilization

PaperPg. 127-142.This study investigates the steady-state and transient response of the squeeze film damper bearing. Both the steady-state and transient equations for the hydrodynamic bearing forces are derived. The steady-state equations are used to determine the damper equivalent stiffness and dampening coefficients. These coefficients are used to find the damper configuration which will provide the optimum support characteristics based on a stability analysis of the rotor-bearing system. The effects of end seals and caveated fluid film are included. The transient analysis of rotor-bearing systems is performed by coupling the dampening and rotor equations and integrating forward in time. The effects of unbalance, cavitation and retainer springs are included in the analysis. Methods of determining the stability of a rotor-bearing system under the influence of aerodynamic forces and internal shaft friction are discussed. Particular emphasis is placed on solving the system characteristic frequency equation and stability maps produced using this method are presented. The study shows that for optimum stability and low force transmissibility the squeeze bearing should operate at an eccentricity ration of e<0.4