Lipid-bound ApoE3 self-assemble into elliptical disc-shaped particles

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE380-255), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE380-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.

Both a Unique Motif at the C Terminus and an N-Terminal HEAT Repeat Contribute to G-Quadruplex Binding and Origin Regulation by the Rif1 Protein

ABSTRACT Rif1 is a key factor for spatiotemporal regulation of DNA replication. Rif1 suppresses origin firing in the mid-late replication domains by generating replication-suppressive chromatin architecture and by recruiting a protein phosphatase. In fission yeast, the function of Hsk1, a kinase important for origin firing, can be bypassed by rif1Δ due to the loss of origin suppression. Rif1 specifically binds to G-quadruplex (G4) in vitro. Here, we show both conserved N-terminal HEAT repeats and C-terminal nonconserved segments are required for origin suppression. The N-terminal 444 amino acids and the C-terminal 229 amino acids can each mediate specific G4 binding, although high-affinity G4 binding requires the presence of both N- and C-terminal segments. The C-terminal 91 amino acids, although not able to bind to G4, can form a multimer. Furthermore, genetic screening led to identification of two classes of rif1 point mutations that can bypass Hsk1, one that fails to bind to chromatin and one that binds to chromatin. These results illustrate functional domains of Rif1 and indicate importance of both the N-terminal HEAT repeat segment and C-terminal G4 binding/oligomerization domain as well as other functionally unassigned segments of Rif1 in regulation of origin firing.

Variation of the CGG Repeat in FMR-1 Gene in Normal and Fragile X Chinese Subjects

The fragile X syndrome is believed to be caused by an expansion of a CGG trinucleotide repeat segment in the FMR-1 gene on the fragile X site of the long arm of the X-chromosome. To understand the variation of the CGG repeat in the FMR-1 gene in southern Chinese from the Hong Kong and Guangzhou area, we undertook the present study. A total of 83 normal and three fragile X subjects were examined. In the normal group, 16 distinct alleles, ranging in size from 272 bp to 332 bp with 17 to 37 CGG repeats were detected. A repeat size of 29 was the most frequent. Compared with data collected in the USA, the repeat size observed in this population was somewhat smaller. Whether this discrepancy is due to ethnic difference remains to be determined. The three fragile X patients examined in this study did not have a greatly expanded CGG segment. One of them may be a mosaic with one full and one premutation allele. The other two patients, although having clinical and cytological features of fragile X syndrome, had a CGG repeat size within normal range. To explain this, we infer that the mutation in these patients may be caused by other mechanisms, such as other types of FMR-1 mutation or mutation in another site. It is possible that the expansion of the CGG repeats may not be as frequent a cause of fragile X syndrome in southern Chinese as in other ethnic groups.

Learning-disabled Males With a Fragile X CGG Expansion in the Upper Premutation Size Range

There is a broad spectrum of clinical involvement in both boys and girls affected by fragile X syndrome. Although this disorder is best known as the most common inherited cause of mental retardation, it also can manifest as learning disabilities in individuals with IQs in the broad range of normal. Boys are usually retarded, and girls are usually learning disabled with fragile X syndrome.1 The responsible gene, fragile X mental retardation 1 (FMR1), was isolated in 1991, and the mutation was found to involve expansion of a trinucleotide (CGG) repeat segment. Individuals with fragile X syndrome have a CGG expansion of more than 200 repeats associated with hypermethylation of both the expansion and an adjacent CpG island (full mutation).2,3

Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations

Tandemly repeated 72-base-pair (bp) segments located between nucleotides 107 and 250 of the simian virus 40 genome are essential for early region transcription. The functional requirement for the 72-bp repeat was supplied even when that segment was translocated to several locations distant from, and in different orientation, relative to, the promoter. Regardless of the position of the 72-bp enhancer segment, transcription was initiated at the same locations as with the normal promoter. Translocation of the 72-bp repeat segment to other sites in the genome resulted in the appearance of DNase I hypersensitivity at that site in the intranuclear viral minichromosomes. One of the translocations which did not produce enhancement of early- and late-region expression also failed to create a DNase I-hypersensitive site at the translocated 72-bp segment.

Simian virus 40 early- and late-region promoter functions are enhanced by the 72-base-pair repeat inserted at distant locations and inverted orientations.

Tandemly repeated 72-base-pair (bp) segments located between nucleotides 107 and 250 of the simian virus 40 genome are essential for early region transcription. The functional requirement for the 72-bp repeat was supplied even when that segment was translocated to several locations distant from, and in different orientation, relative to, the promoter. Regardless of the position of the 72-bp enhancer segment, transcription was initiated at the same locations as with the normal promoter. Translocation of the 72-bp repeat segment to other sites in the genome resulted in the appearance of DNase I hypersensitivity at that site in the intranuclear viral minichromosomes. One of the translocations which did not produce enhancement of early- and late-region expression also failed to create a DNase I-hypersensitive site at the translocated 72-bp segment.