Salvatore Sechi

A multiprotein nuclear complex connects Fanconi anemia and Bloom syndrome.

ABSTRACT Bloom syndrome (BS) is a genetic disorder associated with dwarfism, immunodeficiency, reduced fertility, and an elevated risk of cancer. To investigate the mechanism of this disease, we isolated from human HeLa extracts three complexes containing the helicase defective in BS, BLM. Interestingly, one of the complexes, termed BRAFT, also contains five of the Fanconi anemia (FA) complementation group proteins (FA proteins). FA resembles BS in genomic instability and cancer predisposition, but most of its gene products have no known biochemical activity, and the molecular pathogenesis of the disease is poorly understood. BRAFT displays a DNA-unwinding activity, which requires the presence of BLM because complexes isolated from BLM-deficient cells lack such an activity. The complex also contains topoisomerase IIIα and replication protein A, proteins that are known to interact with BLM and could facilitate unwinding of DNA. We show that BLM complexes isolated from an FA cell line have a lower molecular mass. Our study provides the first biochemical characterization of a multiprotein FA complex and suggests a connection between the BLM and FA pathways of genomic maintenance. The findings that FA proteins are part of a DNA-unwinding complex imply that FA proteins may participate in DNA repair.

The ATRX syndrome protein forms a chromatin-remodeling complex with Daxx and localizes in promyelocytic leukemia nuclear bodies

ATRX syndrome is characterized by X-linked mental retardation associated with α-thalassemia. The gene mutated in this disease, ATRX, encodes a plant homeodomain-like finger and a SWI2/SNF2-like ATPase motif, both of which are often found in chromatin-remodeling enzymes, but ATRX has not been characterized biochemically. By immunoprecipitation from HeLa extract, we found that ATRX is in a complex with transcription cofactor Daxx. The following evidence supports that ATRX and Daxx are components of an ATP-dependent chromatin-remodeling complex: (i) Daxx and ATRX can be coimmunoisolated by antibodies specific for each protein; (ii) a proportion of Daxx cofractionates with ATRX as a complex of 1 MDa by gel-filtration analysis; (iii) in extract from cells of a patient with ATRX syndrome, the level of the Daxx–ATRX complex is correspondingly reduced; (iv) a proportion of ATRX and Daxx colocalize in promyelocytic leukemia nuclear bodies, with which Daxx had previously been located; and (v) the ATRX complex displays ATP-dependent activities that resemble those of other chromatin-remodeling complexes, including triple-helix DNA displacement and alteration of mononucleosome disruption patterns. But unlike the previously described SWI/SNF or NURD complexes, the ATRX complex does not randomize DNA phasing of the mononucleosomes, suggesting that it may remodel chromatin differently. Taken together, the results suggest that ATRX functions in conjunction with Daxx in a novel chromatin-remodeling complex. The defects in ATRX syndrome may result from inappropriate expression of genes controlled by this complex.

The human proteome project: current state and future direction.

After the successful completion of the Human Genome Project, the Human Proteome Organization has recently officially launched a global Human Proteome Project (HPP), which is designed to map the entire human protein set. Given the lack of protein-level evidence for about 30% of the estimated 20,300 protein-coding genes, a systematic global effort will be necessary to achieve this goal with respect to protein abundance, distribution, subcellular localization, interaction with other biomolecules, and functions at specific time points. As a general experimental strategy, HPP research groups will use the three working pillars for HPP: mass spectrometry, antibody capture, and bioinformatics tools and knowledge bases. The HPP participants will take advantage of the output and cross-analyses from the ongoing Human Proteome Organization initiatives and a chromosome-centric protein mapping strategy, termed C-HPP, with which many national teams are currently engaged. In addition, numerous biologically driven and disease-oriented projects will be stimulated and facilitated by the HPP. Timely planning with proper governance of HPP will deliver a protein parts list, reagents, and tools for protein studies and analyses, and a stronger basis for personalized medicine. The Human Proteome Organization urges each national research funding agency and the scientific community at large to identify their preferred pathways to participate in aspects of this highly promising project in a HPP consortium of funders and investigators.

Novel SWI/SNF Chromatin-Remodeling Complexes Contain a Mixed-Lineage Leukemia Chromosomal Translocation Partner

ABSTRACT The SWI/SNF family of chromatin-remodeling complexes has been discovered in many species and has been shown to regulate gene expression by assisting transcriptional machinery to gain access to their sites in chromatin. Several complexes of this family have been reported for humans. In this study, two additional complexes are described that belong to the same SWI/SNF family. These new complexes contain as many as eight subunits identical to those found in other SWI/SNF complexes, and they possess a similar ATP-dependent nucleosome disruption activity. But unlike known SWI/SNFs, the new complexes are low in abundance and contain an extra subunit conserved between human and yeast SWI/SNF complexes. This subunit, ENL, is a homolog of the yeast SWI/SNF subunit, ANC1/TFG3. Moreover, ENL is a fusion partner for the gene product of MLL that is a common target for chromosomal translocations in human acute leukemia. The resultant MLL-ENL fusion protein associates and cooperates with SWI/SNF complexes to activate transcription of the promoter of HoxA7, a downstream target essential for oncogenic activity of MLL-ENL. Our data suggest that human SWI/SNF complexes show considerable heterogeneity, and one or more may be involved in the etiology of leukemia by cooperating with MLL fusion proteins.

The human proteome - A scientific opportunity for transforming diagnostics, therapeutics, and healthcare

A National Institutes of Health (NIH) workshop was convened in Bethesda, MD on September 26–27, 2011, with representative scientific leaders in the field of proteomics and its applications to clinical settings. The main purpose of this workshop was to articulate ways in which the biomedical research community can capitalize on recent technology advances and synergize with ongoing efforts to advance the field of human proteomics. This executive summary and the following full report describe the main discussions and outcomes of the workshop.

The mitochondrial Italian Human Proteome Project initiative (mt-HPP)

Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.

Acrylamide--a cysteine alkylating reagent for quantitative proteomics.

Mass spectrometry-based relative quantification of proteins is often achieved by the labeling of two samples with isotopically light and heavy reagents. The intensities of the ions with different masses, but same chemical properties, can be reliably used for determining relative quantities. Several strategies of labeling with various weakness and strength and degrees of complexity have been described. In this chapter, we describe a simple and inexpensive protein-labeling procedure based on the use of acrylamide and deuterated acrylamide as a cysteine alkylating reagent. Gel electrophoresis is one of the most commonly used techniques for analyzing/visualizing proteins, thus, we emphasize the use of acrylamide as a labeling procedure for quantifying proteins isolated by one- and two-dimensional polyacrylamide gel electrophoresis.

Plasma proteins as early biomarkers of exposure to carcinogenic aromatic amines

Two-dimensional gel electrophoresis (2DG) has been used to study the changes induced in dog plasma polypeptides by the known urinary bladder carcinogens, 4-aminobiphenyl (4-ABP) and 2-naphthylamine (2-NA). Treatment with 3-aminobiphenyl (3-ABP) and 1-naphthylamine (1-NA), both considered to be non-carcinogenic, were used as controls. The purpose of this study was: (1) to determine whether or not changes that occurred in the plasma protein patterns were specific to 4-ABP and/or other related carcinogenic arylamines; (2) to measure the time course in the changes of the major polypeptides during dosing and their resynthesis during a recovery period; and (3) to determine, by microsequencing, the biochemical identity of the affected proteins. The results indicate that only the most potent carcinogen, 4-ABP, had the effect of suppressing the expression of some proteins, while the other aromatic amines caused no discernible change in the 2DG patterns during a 12-week dosing period. The 4-ABP caused dramatic suppression of two sets of proteins. One set of three spots had an apparent molecular weight of 32.5 kDa, and a pI of 5.8-6.0. The major component in this group was identified as the beta-chain of haptoglobin. Expression of this protein decreased markedly during the first 2 weeks of treatment and recovered slowly after dosing stopped. Since haptoglobin functions to bind with free hemoglobin and facilitates its elimination from the blood stream, these results can be rationalized as a consequence of 4-ABP binding to hemoglobin in the erythrocyte, resulting in cell death and hemolysis. The 4-ABP modified hemoglobin then binds to haptoglobin and this tertiary complex is purged from the blood stream, resulting in the disappearance of free haptoglobin. A second set of spots (mol. wt., 65 kDa; pI, 6.5-6.6) disappeared much faster than the haptoglobin, and recovered more quickly. The major protein is about one-fifth the intensity of haptoglobin and appeared to be N-terminally blocked. Internal microsequencing of four fragments obtained from tryptic cleavage of the major spot of this group showed significant similarity to the serum albumin sequence of several species. This spot group is not the major serum albumin spot, however, since the latter is readily identified as the most abundant spot on the plasma map. During the course of this study, several other polypeptides in the 2DG map of dog plasma were identified and are presented here.