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Biomedical subjects

Alma L Burlingame

Publications and source records attributed to Alma L Burlingame.

11 recordsLinked to original sources
PubMed2026

Druggable genome CRISPRi screen in hydrogels reveals regulators of cortactin-driven actin remodeling promoting glioblastoma invasion.

To identify therapeutic targets limiting glioblastoma invasion, we applied druggable genome CRISPRi screens and multiomic analysis to patient-derived glioblastoma cells in micro-dissectible biomimetic 3D hydrogels that permitted separation and analysis of core versus invasive fractions. Of 2,550 genes screened, 12 encoded druggable targets whose suppression limited invasion, of which AURKB (encoding aurora kinase B) and ACP1 (encoding low molecular weight protein tyrosine phosphatase, LMW-PTP) were validated in neurosphere assays and in vivo. Proximity labeling identified cortactin as a link between LMW-PTP and aurora B, and we observed that cortactin underwent serine phosphorylation by aurora B and tyrosine dephosphorylation by LMW-PTP. Targeting ACP1 or AURKB via CRISPRi or inhibitors in culture and in vivo shifted the cortactin phosphorylation balance in glioblastoma, reducing levels of cortactin and the actin-related protein 2/3 (Arp2/3) complex that mediates cortactin-induced actin stabilization, thereby reducing actin-cortactin-Arp2/3 colocalization and subsequent actin polymerization. AURKB or ACP1 targeting shifted actin from cytoplasm to the nucleus, reducing mesenchymal gene expression. Biophysical analysis implicated AURKB in glioblastoma cell adhesion and stiffness needed for initial migration and ACP1 in mechanical stress resistance required for later migration. These findings revealed a targetable axis balancing kinase and phosphatase activities to regulate actin polymerization during glioblastoma invasion.

Humans↗
PubMed2025

Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks.

Loss-of-function variants in TBK1, encoding a protein kinase, are strongly associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how haploinsufficiency for TBK1 leads to age-related neurodegeneration remains unresolved. Here, we utilize sets of isogenic induced pluripotent stem cells (iPSCs) with loss of TBK1 or loss of optineurin (OPTN) for quantitative global proteomics and phospho-proteomics in both stem cells and excitatory neurons. We found that TBK1 sustains the abundance and phosphorylation of its interacting adapter proteins, AZI2/NAP1, TANK, and TBKBP1/SINTBAD. Moreover, TBK1 regulates the phosphorylation of endo-lysosomal proteins, such as GABARAPL2, the late-endosome GTPase RAB7A, and selective autophagy cargo receptor proteins-including novel phospho-sites in p62/SQSTM1-in neurons. Finally, we provide a census of the phospho-proteome in nascent human neurons for further studies. Overall, TBK1 serves as a point of convergence in ALS/FTD-linked endo-lysosomal networks that act in a cell-autonomous manner to maintain protein homeostasis in neurons.

Humans↗
PubMed2003

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Amino Acid Sequence↗
PubMed2003

Detection and analysis of urinary peptides by on-line liquid chromatography and mass spectrometry: application to patients with renal Fanconi syndrome.

Urinary proteomics has become a topical and potentially valuable field of study in relation to normal and abnormal renal function. Filtered bioactive peptides present in high concentration in the nephron of patients with tubular proteinuria may have downstream effects on renal tubular function. In renal Fanconi syndromes, such as Dent's disease, peptides implicated in altered tubular function or injury have recently been measured in urine by immunochemical methods. However, the limited availability of antibodies means that only certain peptides can be detected in this way. We have used nanoflow liquid chromatography and tandem mass spectrometry (nanoLC-MS/MS) as a complementary technique to analyse urinary peptides. Urine was desalted by solid-phase extraction (SPE) and its peptides were then separated from neutral and acidic compounds by strong cation-exchange chromatography (SCX), which was also used to fractionate the peptide mixture. Fractions from the SCX step were separated further by reversed-phase LC and analysed on-line by MS/MS. Extraction by SPE showed a good recovery of small peptides. We detected over 100 molecular species in urine samples from three individuals with Dent's disease. In addition to plasma and known urinary proteins, we identified some novel proteins and potentially bioactive peptides in urine from these patients, which were not present in normal urine. These data show that nanoLC-MS/MS complements existing techniques for the identification of polypeptides in urine. This approach is a potentially powerful tool to discover new markers and/or causative factors in renal disease; in addition, its sensitivity may also make it applicable to the direct ultramicroanalysis of renal tubule fluid.

Biomarkers↗
PubMed2002

Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry.

A new strategy has been employed for the identification of the covalent modification sites (mainly acetylation and methylation) of histone H3 from chicken erythrocytes using low enzyme/substrate ratios and short digestion times (trypsin used as the protease) with analysis by HPLC separation, matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF), matrix-assisted laser desorption ionization-postsource decay, and tandem mass spectrometric techniques. High-accuracy MALDI-TOF mass measurements with representative immonium ions (126 for acetylated lysine, 98 for monomethylated lysine, and 84 for di-, tri-, and unmethylated lysine) have been effectively used for differentiating methylated peptides from acetylated peptides. Our results demonstrate that lysines 4, 9, 14, 27, and 36 of the N-terminal of H3 are methylated, while lysines 14, 18, and 23 are acetylated. Surprisingly, a non-N-terminal residue, lysine 79, in the loop region hooking up to the bound DNA, was newly found to be methylated (un-, mono-, and dimethylated isoforms coexist). The reported mass spectrometric method has the advantages of speed, directness, sensitivity, and ease over protein sequencing and Western-blotting methods and holds the promise of an improved method for determining the status of histone modifications in the field of chromosome research.

Acetylation↗
PubMed2002

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization.

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.

Amino Acid Sequence↗
PubMed2002

Differential proteome analysis of replicative senescence in rat embryo fibroblasts.

Normal somatic cells undergo a finite number of divisions and then cease dividing whereas cancer cells are able to proliferate indefinitely. To identify the underlying mechanisms that limit the mitotic potential, a two-dimensional differential proteome analysis of replicative senescence in serially passaged rat embryo fibroblasts was undertaken. Triplicate independent two-dimensional gels containing over 1200 spots each were run, curated, and analyzed. This revealed 49 spots whose expression was altered more than 2-fold. Of these, 42 spots yielded positive protein identification by mass spectrometry comprising a variety of cytoskeletal, heat shock, and metabolic proteins, as well as proteins involved in trafficking, differentiation, and protein synthesis, turnover, and modification. These included gelsolin, a candidate tumor suppressor for breast cancer, and alpha-glucosidase II, a member of the family of glucosidases that includes klotho; a defect in klotho expression in mice results in a syndrome that resembles human aging. Changes in expression of TUC-1, -2, -4, and -4 beta, members of the TUC family critical for neuronal differentiation, were also identified. Some of the identified changes were also shown to occur in two other models of senescence, premature senescence of REF52 cells and replicative senescence of mouse embryo fibroblasts. The majority of these candidate proteins were unrecognized previously in replicative senescence. They are now implicated in a new role.

Animals↗
PubMed2002

The identification of protein-protein interactions of the nuclear pore complex of Saccharomyces cerevisiae using high throughput matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry.

Mass spectrometry has become the technology of choice for detailed identification of proteins in complex mixtures. Although electrophoretic separation, proteolytic digestion, mass spectrometric analysis of unseparated digests, and database searching have become standard methods in widespread use, peptide sequence information obtained by collision-induced dissociation and tandem mass spectrometry is required to establish the most comprehensive and reliable results. Most tandem mass spectrometers in current use employ electrospray ionization. In this work a novel tandem mass spectrometer employing matrix-assisted laser desorption ionization-time-of-flight/time-of-flight operating at 200 Hz has been used to identify proteins interacting with known nucleoporins in the nuclear pore complex of Saccharomyces cerevisiae. Proteins interacting with recombinant proteins as bait were purified from yeast extracts and then separated by one-dimensional SDS-PAGE. Although peptide mass fingerprinting is sometimes sufficient to identify proteins, this study shows the importance of employing tandem mass spectrometry for identifying proteins in mixtures or as covalently modified forms. The rules for incorporating these features into MS-Tag are presented. In addition to providing an evaluation of the sensitivity and overall quality of collision-induced dissociation spectra obtained, standard conditions for ionization and fragmentation have been selected that would allow automatic data collection and analysis, without the need to adjust parameters in a sample-specific fashion. Other considerations essential for successful high throughput protein analysis are discussed.

Amino Acid Sequence↗
PubMed2002

Histone acetylation and deacetylation: identification of acetylation and methylation sites of HeLa histone H4 by mass spectrometry.

The acetylation isoforms of histone H4 from butyrate-treated HeLa cells were separated by C(4) reverse-phase high pressure liquid chromatography and by polyacrylamide gel electrophoresis. Histone H4 bands were excised and digested in-gel with the endoprotease trypsin. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry was used to characterize the level of acetylation, and nanoelectrospray tandem mass spectrometric analysis of the acetylated peptides was used to determine the exact sites of acetylation. Although there are 15 acetylation sites possible, only four acetylated peptide sequences were actually observed. The tetra-acetylated form is modified at lysines 5, 8, 12, and 16, the tri-acetylated form is modified at lysines 8, 12, and 16, and the di-acetylated form is modified at lysines 12 and 16. The only significant amount of the mono-acetylated form was found at position 16. These results are consistent with the hypothesis of a "zip" model whereby acetylation of histone H4 proceeds in the direction of from Lys-16 to Lys-5, and deacetylation proceeds in the reverse direction. Histone acetylation and deacetylation are coordinated processes leading to a non-random distribution of isoforms. Our results also revealed that lysine 20 is di-methylated in all modified isoforms, as well as the non-acetylated isoform of H4.

Acetylation↗
PubMed2002

Structure and functional characterization of the periplasmic N-terminal polypeptide domain of the sugar-specific ion channel protein (ScrY porin).

The structure of the sucrose-specific porin (ScrY) from Salmonella typhimurium has been elucidated by X-ray crystallography to consist of 18 antiparallel beta-strands, associated as a trimer complex similar to ion-transport channels. However, the 71-amino-acid-residue N-terminal periplasmic domain was not determined from the crystal structure due to the absence of sufficient electron density. The N-terminal polypeptide contains a coiled-coil structural motif and has been assumed to play a role in the sugar binding of ScrY porin. In this study the proteolytic stability and a specific proteolytic truncation site at the N-terminal domain were identified by the complete primary structure characterization of ScrY porin, using MALDI mass spectrometry and post-source-decay fragmentation. The secondary structure and supramolecular association of the coiled-coil N-terminal domain were determined by chemical synthesis of the complete N-terminal polypeptide and several partial sequences and their spectroscopic, biophysical, and mass spectrometric characterization. Circular dichroism spectra revealed predominant alpha-helical conformation for the putative coiled-coil domain comprising residues 4-46. Specific association to both dimer and trimer complexes was identified by electrospray ionization mass spectra and was ascertained by dynamic light scattering and electrophoresis data. The role of the N-terminal domain in sugar binding was examined by comparative TR-NOE-NMR spectroscopy of the complete ScrY porin and a recombinant mutant, ScrY(delta1-62), lacking the N-terminal polypeptide. The TR-NOE-NMR data showed a strong influence of ScrY porin on the sugar-binding affinity and suggested a possible function of the periplasmic N terminus for supramolecular stabilization and low-affinity sugar binding.

Amino Acid Sequence↗