Javier A. Alfaro

Hotspot activating PRKD1 somatic mutations in polymorphous low-grade adenocarcinomas of the salivary glands

Polymorphous low-grade adenocarcinoma (PLGA) is the second most frequent type of malignant tumor of the minor salivary glands. We identified PRKD1 hotspot mutations encoding p.Glu710Asp in 72.9% of PLGAs but not in other salivary gland tumors. Functional studies demonstrated that this kinase-activating alteration likely constitutes a driver of PLGA.

ABO(H) blood group A and B glycosyltransferases recognize substrate via specific conformational changes.

The final step in the enzymatic synthesis of the ABO(H) blood group A and B antigens is catalyzed by two closely related glycosyltransferases, an α-(1→3)-N-acetylgalactosaminyltransferase (GTA) and an α-(1→3)-galactosyltransferase (GTB). Of their 354 amino acid residues, GTA and GTB differ by only four “critical” residues. High resolution structures for GTB and the GTA/GTB chimeric enzymes GTB/G176R and GTB/G176R/G235S bound to a panel of donor and acceptor analog substrates reveal “open,” “semi-closed,” and “closed” conformations as the enzymes go from the unliganded to the liganded states. In the open form the internal polypeptide loop (amino acid residues 177-195) adjacent to the active site in the unliganded or H antigen-bound enzymes is composed of two α-helices spanning Arg180-Met186 and Arg188-Asp194, respectively. The semi-closed and closed forms of the enzymes are generated by binding of UDP or of UDP and H antigen analogs, respectively, and show that these helices merge to form a single distorted helical structure with alternating α-310-α character that partially occludes the active site. The closed form is distinguished from the semi-closed form by the ordering of the final nine C-terminal residues through the formation of hydrogen bonds to both UDP and H antigen analogs. The semi-closed forms for various mutants generally show significantly more disorder than the open forms, whereas the closed forms display little or no disorder depending strongly on the identity of residue 176. Finally, the use of synthetic analogs reveals how H antigen acceptor binding can be critical in stabilizing the closed conformation. These structures demonstrate a delicately balanced substrate recognition mechanism and give insight on critical aspects of donor and acceptor specificity, on the order of substrate binding, and on the requirements for catalysis.

Onco-proteogenomics: cancer proteomics joins forces with genomics

The complexities of tumor genomes are rapidly being uncovered, but how they are regulated into functional proteomes remains poorly understood. Standard proteomics workflows use databases of known proteins, but these databases do not capture the uniqueness of the cancer transcriptome, with its point mutations, unusual splice variants and gene fusions. Onco-proteogenomics integrates mass spectrometry–generated data with genomic information to identify tumor-specific peptides. Linking tumor-derived DNA, RNA and protein measurements into a central-dogma perspective has the potential to improve our understanding of cancer biology.

Glycosyltransferase Structure and Function

The biosynthesis of the oligosaccharides and polysaccharides observed in any organism requires the existence of a repertoire of glycosyltransferase enzymes that catalyze the sequential transfer of sugars from a specific activated donor to a specific acceptor molecule to form regio- and stereospecific glycosidic linkages. A viral genome may encode just one glycosyltransferase, while a mammalian genome encodes hundreds of these enzymes. It is notable that approximately 1% of open reading frames over all sequenced genomes have been found to be glycosyltransferases, which is a fraction comparable to that allotted to kinases. Glycosyltransferases are a highly diverse group of enzymes with little homology even among enzymes that share the same substrate specificity. Classification of glycosyltransferases according to sequence homology reveals at least 86 families; however, to date only 27 of these families have members with known structure. This is in sharp contrast with glycosylhydrolases, which to date have published structures for 70 of the so far described 102 classes. The paucity of structural data for glycosyltransferases has been attributed to their membrane-associated character and low expression levels, but even with the relatively limited number of available structures it is possible to see emerging trends that offer a glimpse of the principles of enzyme structure.

Aberrant DNA methylation reprogramming during induced pluripotent stem cell generation is dependent on the choice of reprogramming factors

The conversion of somatic cells into pluripotent stem cells via overexpression of reprogramming factors involves epigenetic remodeling. DNA methylation at a significant proportion of CpG sites in induced pluripotent stem cells (iPSCs) differs from that of embryonic stem cells (ESCs). Whether different sets of reprogramming factors influence the type and extent of aberrant DNA methylation in iPSCs differently remains unknown. In order to help resolve this critical question, we generated human iPSCs from a common fibroblast cell source using either the Yamanaka factors (OCT4, SOX2, KLF4 and cMYC) or the Thomson factors (OCT4, SOX2, NANOG and LIN28), and determined their genome-wide DNA methylation profiles. In addition to shared DNA methylation aberrations present in all our iPSCs, we identified Yamanaka-iPSC (Y-iPSC)-specific and Thomson-iPSC (T-iPSC)-specific recurrent aberrations. Strikingly, not only were the genomic locations of the aberrations different but also their types: reprogramming with Yamanaka factors mainly resulted in failure to demethylate CpGs, whereas reprogramming with Thomson factors mainly resulted in failure to methylate CpGs. Differences in the level of transcripts encoding DNMT3b and TET3 between Y-iPSCs and T-iPSCs may contribute partially to the distinct types of aberrations. Finally, de novo aberrantly methylated genes in Y-iPSCs were enriched for NANOG targets that are also aberrantly methylated in some cancers. Our study thus reveals that the choice of reprogramming factors influences the amount, location, and class of DNA methylation aberrations in iPSCs. These findings may provide clues into how to produce human iPSCs with fewer DNA methylation abnormalities.

Sequence-dependent effects of cryoprotectants on the active sites of the human ABO(H) blood group A and B glycosyltransferases.

The human ABO(H) A and B blood group glycosyltransferases GTA and GTB differ by only four amino acids, yet this small dissimilarity is responsible for significant differences in biosynthesis, kinetics and structure. Like other glycosyltransferases, these two enzymes have been shown to recognize substrates through dramatic conformational changes in mobile polypeptide loops surrounding the active site. Structures of GTA, GTB and several chimeras determined by single-crystal X-ray diffraction demonstrate a range of susceptibility to the choice of cryoprotectant, in which the mobile polypeptide loops can be induced by glycerol to form the ordered closed conformation associated with substrate recognition and by MPD [hexylene glycol, (±)-2-methyl-2,4-pentanediol] to hinder binding of substrate in the active site owing to chelation of the Mn²⁺ cofactor and thereby adopt the disordered open state. Glycerol is often avoided as a cryoprotectant when determining the structures of carbohydrate-active enzymes as it may act as a competitive inhibitor for monosaccharide ligands. Here, it is shown that the use of glycerol as a cryoprotectant can additionally induce significant changes in secondary structure, a phenomenon that could apply to any class of protein.

Detecting protein variants by mass spectrometry: a comprehensive study in cancer cell-lines

BackgroundOnco-proteogenomics aims to understand how changes in a cancer’s genome influences its proteome. One challenge in integrating these molecular data is the identification of aberrant protein products from mass-spectrometry (MS) datasets, as traditional proteomic analyses only identify proteins from a reference sequence database.MethodsWe established proteomic workflows to detect peptide variants within MS datasets. We used a combination of publicly available population variants (dbSNP and UniProt) and somatic variations in cancer (COSMIC) along with sample-specific genomic and transcriptomic data to examine proteome variation within and across 59 cancer cell-lines.ResultsWe developed a set of recommendations for the detection of variants using three search algorithms, a split target-decoy approach for FDR estimation, and multiple post-search filters. We examined 7.3 million unique variant tryptic peptides not found within any reference proteome and identified 4771 mutations corresponding to somatic and germline deviations from reference proteomes in 2200 genes among the NCI60 cell-line proteomes.ConclusionsWe discuss in detail the technical and computational challenges in identifying variant peptides by MS and show that uncovering these variants allows the identification of druggable mutations within important cancer genes.

Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine

Cells secrete biomolecules into the extracellular space as a way of intercellular communication. Secreted proteins can act as ligands that engage specific receptors—on the same cell, nearby cells, or distant cells—and induce defined signaling pathways. Proteins and other biomolecules can also be packaged as cargo molecules within vesicles that are released to the extracellular space (termed extracellular vesicles or EVs). A subclass of such EVs, exosomes have been shown to horizontally transfer information. In recent years, exosomes have sparked tremendous interest in biological research, both for the discovery of novel biomarkers and for the identification of signaling molecules, as part of their cargo. Although multiple methods have been described for the isolation of exosomes, described here is a simple differential centrifugation approach that is well suited for the isolation of exosomes from conditioned cell culture media and urine. Mass spectrometry provides an ideal method to comprehensively analyze the protein cargo of exosomes.

Proteomic Profiling of Secreted Proteins, Exosomes, and Microvesicles in Cell Culture Conditioned Media

Secreted proteins are of tremendous biological interest since they can act as ligands for receptors to activate downstream signalling cascades or be used as biomarkers if altered abundance is correlated with a specific pathological state. Proteins can be secreted either as soluble molecules or as part of extracellular vesicles (i.e., exosomes or microvesicles). The complete proteomic profiling of secretomes requires analysis of secreted proteins and extracellular vesicles. Hence, the method described here enriches for microvesicles, exosomes, and secreted proteins from conditioned media using differential centrifugation. The three fractions are then analyzed by mass spectrometry-based proteomics for in-depth characterization and comparison of the protein secretome of cell lines.