The Mechanostability of Isolated Focal Adhesions Is Strongly Dependent on pH

SummaryThis report demonstrates that the mechanical stability of focal adhesions exhibits a biphasic and sensitive pH dependence. These studies used isolated focal adhesions, which retain many of the properties of the intracellular structures, including protein composition and force-dependent reinforcement by cytosolic proteins. The focal adhesion structures are least stable to applied force at a pH of 6.4, and significantly more stable at slightly higher and lower pH values. This trend is consistent with previous work that characterized the pH dependence of cell migration and may therefore be relevant to controlling the invasiveness of metastatic cancer cells. This approach is significant because it allows biochemical studies of large protein complexes previously studied only in cell culture, and therefore offers new opportunities for performing mechanistic studies of a range of factors that contribute to focal adhesion stability

The Activity of HDAC8 Depends on Local and Distal Sequences of Its Peptide Substrates†

ABSTRACT: This paper introduces a flexible assay for characterizing the activities of the histone deacetylase enzymes. The approach combines mass spectrometry with self-assembled monolayers that present acetylated peptides and enables a label-free and one-step assay of this biochemical activity. The assay was used to characterize the activity of HDAC8 toward peptides taken from the N-terminal tail of the H4 histone and reveals that a distal region of the peptide substrate interacts with the deacetylase at an exosite and contributes to the activity of the substrate. Specifically, a peptide corresponding to residues 8-19 of H4 and having lysine 12 acetylated is an active substrate, but removal of the KRHR (residues 16-19) sequence abolishes activity. Mutation of glycine 11 to arginine in the peptide lacking the KRHR sequence restores activity, demonstrating that both local and distal sequences act synergistically to regulate the activity of the HDAC. Assays with peptides bearing multiply acetylated residues, but in which each acetyl group is isotopically labeled, permit studies of the processive deacetylation of peptides. Peptide substrates having an extended sequence that includes K20 were used to demonstrate that methylation of this residue directly affects HDAC8 activity at K12. This work provides a mechanistic basis for the regulation of HDAC activities by distal sequences and may contribute to studies aimed at evaluating the role of the histone code in regulating gene expression

Combining microfluidic networks and peptide arrays for multi-enzyme assays

This paper reports the use of microfluidic networks (μFNs) to both prepare peptide microarrays and carry out label-free enzyme assays on self-assembled monolayers (SAMs) of alkanethiolates on gold. A poly(dimethylsiloxane) (PDMS) stamp fabricated with microchannels is used to immobilize a linear array of cysteine-terminated peptides onto SAMs presenting maleimide groups. The stamp is then reapplied to the SAM in a perpendicular direction to introduce enzyme solutions so that each solution can interact with an identical linear array of immobilized peptides. The μFNs enable multiple enzyme−substrate interactions to be simultaneously evaluated at a submicroliter scale, while the use of SAMs enables the use of MALDI mass spectrometry (MS) to analyze the enzyme activities. This paper demonstrates applications of this system for assaying multiple kinases and for profiling the activities of kinases and phosphatases in human K562 cell extracts. The combination of μFN, SAMs, and MS detection provides a flexible platform for assaying enzyme activities in biological samples

The Synergy Peptide PHSRN and the Adhesion Peptide RGD Mediate Cell Adhesion through a Common Mechanism†

ABSTRACT: This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin R5 or anti-integrin â1 antibody. This work demonstrates unambiguously that PHSRN alone ca

Attachment of cells to islands presenting gradients of adhesion ligands

This paper reports a strategy that uses microfluidic networks to pattern self-assembled monolayers with gradient microislands for the attachment of individual cells. A microfluidic network is first used to pattern a monolayer into square regions that present maleimide groups and then used to flow a solution having a gradient of the cell adhesion peptide Arg-Gly-Asp over the substrate. In this way, the surface is patterned with microislands approximately 33 x 33 micrometers in size and each having a defined gradient of immobilized cell adhesion ligand. B16F10 cells were allowed to attach to the patterned islands and were found to display a nonuniform distribution of cytoskeletal structures in response to the gradient of adhesion ligand. This work is significant because it permits studies of the influence of a nonuniform microenvironment on the polarization, differentiation, and signaling of adherent cells

High Throughput Screening with SAMDI Mass Spectrometry for Directed Evolution

Advances in directed evolution have led to an exploration of new and important chemical transformations; however, many of these efforts still rely on the use of low-throughput chromatography-based screening methods. We present a high-throughput strategy for screening libraries of enzyme variants for improved activity. Unpurified reaction products are immobilized to a self-assembled monolayer and analyzed by mass spectrometry, allowing for direct evaluation of thousands of variants in under an hour. The method was demonstrated with libraries of randomly mutated cytochrome P411 variants to identify improved catalysts for C–H alkylation. The technique may be tailored to evolve enzymatic activity for a variety of transformations where higher throughput is needed

Structural and dynamic characterization of the heterodimeric and homodimeric complexes of distamycin and 1-methylimidazole-2-carboxamide-netropsin bound to the minor groove of DNA

NMR spectroscopy combined with molecular modeling was used to characterize a heterodimeric complex with Dst and 2-ImN bound in the minor groove of d(GCCTAACAAGG)•d(CCTTGTTAGGC) (1:1:1 2-ImN•Dst•DNA complex). The imidazole-pyrrole-pyrrole ligand 2-ImN spans 5'-GTTA-3' of the TAACA•TGTTA binding site with the imidazole nitrogen specifically recognizing the guanine amino group. The Dst ligand lies along the 5'-AACA-3' sequence and complements the 2-ImN ligand in the formation of the antiparallel side-by-side heterodimeric complex. Titrations of the same site with Dst or 2-ImN alone yield homodimeric complexes (2:1 ligand.DNA) of lower stability than the 1:1:1 2-ImN•Dst•DNA complex. Dst and 2-ImN binding to d(CGCAAACTGGC)•d(GCCAGTTTGCG) was also investigated. The 1:1:1 2-ImN•Dst•DNA complex is again the most stable complex with the AAACT•AGTTT site and is similar to the TAACA•TGTTA complex. No monomeric binding of either 2-ImN or Dst was observed to either site

Peptide Arrays Identify Isoform-Selective Substrates for Profiling Endogenous Lysine Deacetylase Activity

A cetylation of lysine is a post-translational modifi-cation involved inmany eukaryotic cellular pro-cesses (1). Though most commonly associated with histone proteins (2), a recent proteomic study by Mann and colleagues identified nearly 2,000 proteins in acetylated form and showed that this modification is used globally in regulating cell function (3). This recogni-tion hasmotivated a renaming of the histone acetyltrans-ferase (HAT) and histone deacetylase (HDAC) enzymes to reflect their broader roles [now the lysine acetyltrans-ferases (KATs) and the lysine deacetylases (KDACs) (4)] and has highlighted our limited understanding of the functions of the individual enzymes responsible for achieving andmaintaining acetylation states. A significant effort is now directed at understanding the differential roles that the 18 human KDAC isoforms play in regulating cell behavior (5−8). These studies re