The AAA+ protein ATAD3 has displacement loop binding properties and is involved in mitochondrial nucleoid organization.

Many copies of mammalian mitochondrial DNA contain a short triple-stranded region, or displacement loop (D-loop), in the major noncoding region. In the 35 years since their discovery, no function has been assigned to mitochondrial D-loops. We purified mitochondrial nucleoprotein complexes from rat liver and identified a previously uncharacterized protein, ATAD3p. Localization studies suggested that human ATAD3 is a component of many, but not all, mitochondrial nucleoids. Gene silencing of ATAD3 by RNA interference altered the structure of mitochondrial nucleoids and led to the dissociation of mitochondrial DNA fragments held together by protein, specifically, ones containing the D-loop region. In vitro, a recombinant fragment of ATAD3p bound to supercoiled DNA molecules that contained a synthetic D-loop, with a marked preference over partially relaxed molecules with a D-loop or supercoiled DNA circles. These results suggest that mitochondrial D-loops serve to recruit ATAD3p for the purpose of forming or segregating mitochondrial nucleoids.

Amyloid-beta as a "difficult sequence" in solid phase peptide synthesis.

The phenomenon of "difficult sequence" has long frustrated chemists in their efforts to assemble peptides that contain such sequences by solid phase synthesis methods. A variety of remedial measures are available to minimize or even abolish the negative impact of these sequences during synthesis. These include the use of elevated temperatures and stronger acylating reagents. Amyloid-beta, a fragment of the amyloid precursor protein, contains 40-43 residues and possesses a C-terminal sequence that is particularly resistant to ready solid phase synthesis making it a "difficult sequence" peptide. This review focuses on approaches to successfully assemble the peptide by both Boc- and Fmoc solid phase synthesis.

Application of 2-D free-flow electrophoresis/RP-HPLC for proteomic analysis of human plasma depleted of multi high-abundance proteins.

Free-flow electrophoresis (FFE) and rapid (6 min) RP-HPLC was used to fractionate human citrate-treated plasma. Prior to analysis, the six most abundant proteins in plasma were removed by immunoaffinity chromatography; both depleted plasma and the fraction containing the six abundant proteins depleted were taken for MS-based analysis. Fractionated proteins were digested with trypsin and the generated peptides were subjected to MS-based peptide sequencing. To date, 78 plasma proteins have been unambiguously identified by manual validation from 16% (15/96 FFE total fractions) of the collected FFE pools; 55 identifications were based on > or = 2 tryptic peptides and 23 using single peptides. The molecular weight range of proteins and peptides isolated by this method ranged from approximately 190 K (e.g., Complement C3 and C4) to approximately 4-6 K (e.g., CRISPP and Apolipoprotein C1). This FFE/RP-HPLC approach reveals low-abundance proteins and peptides (e.g., L-Selectin approximately 17 ng/mL and the cancer-associated SCM-recognition, immunodefense suppression, and serine protease protection peptide (CRISPP) at approximately 0.5-1 ng/mL), where CRISPP was found in association with alpha-1-antitrypsin as a non-covalent complex, in the fraction containing the depleted high-abundance proteins. In contrast to shotgun proteomic approaches, the FFE/RP-HPLC method described here allows the identification of potentially interesting peptides to be traced back to their protein of origin, and for the first time, has confirmed the "protein sponge" hypothesis where the 35 residue CRISPP polypeptide is non-covalently complexed with the major circulating plasma protein alpha-1-antitrypsin.