Proteome-wide remodeling of protein location and function by stress.

Protein location and function can change dynamically depending on many factors, including environmental stress, disease state, age, developmental stage, and cell type. Here, we describe an integrative computational framework, called the conditional function predictor (CoFP; http://nbm.ajou.ac.kr/cofp/), for predicting changes in subcellular location and function on a proteome-wide scale. The essence of the CoFP approach is to cross-reference general knowledge about a protein and its known network of physical interactions, which typically pool measurements from diverse environments, against gene expression profiles that have been measured under specific conditions of interest. Using CoFP, we predict condition-specific subcellular locations, biological processes, and molecular functions of the yeast proteome under 18 specified conditions. In addition to highly accurate retrieval of previously known gold standard protein locations and functions, CoFP predicts previously unidentified condition-dependent locations and functions for nearly all yeast proteins. Many of these predictions can be confirmed using high-resolution cellular imaging. We show that, under DNA-damaging conditions, Tsr1, Caf120, Dip5, Skg6, Lte1, and Nnf2 change subcellular location and RNA polymerase I subunit A43, Ino2, and Ids2 show changes in DNA binding. Beyond specific predictions, this work reveals a global landscape of changing protein location and function, highlighting a surprising number of proteins that translocate from the mitochondria to the nucleus or from endoplasmic reticulum to Golgi apparatus under stress.

The ß-1,3-glucanosyltransferase Gas1 regulates Sir2-mediated rDNA stability in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the stability of highly repetitive rDNA array is maintained through transcriptional silencing. Recently, a ß-1,3-glucanosyltransferase Gas1 has been shown to play a significant role in the regulation of transcriptional silencing in S. cerevisiae. Here, we show that the gas1Δ mutation increases rDNA silencing in a Sir2-dependent manner. Remarkably, the gas1Δ mutation induces nuclear localization of Msn2/4 and stimulates the expression of PNC1, a gene encoding a nicotinamidase that functions as a Sir2 activator. The lack of enzymatic activity of Gas1 or treatment with a cell wall-damaging agent, Congo red, exhibits effects similar to those of the gas1Δ mutation. Furthermore, the loss of Gas1 or Congo red treatment lowers the cAMP-dependent protein kinase (PKA) activity in a cell wall integrity MAP kinase Slt2-dependent manner. Collectively, our results suggest that the dysfunction of Gas1 plays a positive role in the maintenance of rDNA integrity by decreasing PKA activity and inducing the accumulation of Msn2/4 in the nucleus. It seems that nuclear-localized Msn2/4 stimulate the expression of Pnc1, thereby enhancing the association of Sir2 with rDNA and promoting rDNA stability.

Improving brush polymer infrared one-dimensional photonic crystals via linear polymer additives.

Brush block copolymers (BBCPs) enable the rapid fabrication of self-assembled one-dimensional photonic crystals with photonic band gaps that are tunable in the UV-vis-IR, where the peak wavelength of reflection scales with the molecular weight of the BBCPs. Due to the difficulty in synthesizing very large BBCPs, the fidelity of the assembled lamellar nanostructures drastically erodes as the domains become large enough to reflect IR light, severely limiting their performance as optical filters. To overcome this challenge, short linear homopolymers are used to swell the arrays to ∼180% of the initial domain spacing, allowing for photonic band gaps up to ∼1410 nm without significant opacity in the visible, demonstrating improved ordering of the arrays. Additionally, blending BBCPs with random copolymers enables functional groups to be incorporated into the BBCP array without attaching them directly to the BBCPs. The addition of short linear polymers to the BBCP arrays thus offers a facile means of improving the self-assembly and optical properties of these materials, as well as adding a route to achieving films with greater functionality and tailorability, without the need to develop or optimize the processing conditions for each new brush polymer synthesized.

How water layers on graphene affect folding and adsorption of TrpZip2.

We present a computational study of the folding of the Trp-rich ß-hairpin TrpZip2 near graphene, a surface of interest as a platform for biosensors. The protein adsorbs to the surface, populating a new bound, folded state, coexisting with extended, adsorbed conformations. Adsorption and folding are modulated by direct interactions between the indole rings of TrpZip2 and the rings on the graphene surface, as well as by indirect water-mediated interactions. In particular, we observe strong layering of water near graphene, ice-like water configurations, and the formation of short lived hydrogen-bonds between water and protein. In order to study the effect of this layering in more detail, we modified the interactions between graphene and water to obtain two extreme cases: (1) enhanced layering of water that prevents the peptide from penetrating the water layer thereby enabling it to fold to a bulk-like structure, and (2) disruption of the water layer leading to adsorption and unfolding of the protein on the surface. These studies illuminate the roles of direct and solvent mediated interactions in modulating adsorption and folding of proteins on surfaces.

Atg1-dependent phosphorylation of Vps34 is required for dynamic regulation of the phagophore assembly site and autophagy in Saccharomyces cerevisiae.

Macroautophagy/autophagy is a key catabolic pathway in which double-membrane autophagosomes sequester various substrates destined for degradation, enabling cells to maintain homeostasis and survive under stressful conditions. Several autophagy-related (Atg) proteins are recruited to the phagophore assembly site (PAS) and cooperatively function to generate autophagosomes. Vps34 is a class III phosphatidylinositol 3-kinase, and Atg14-containing Vps34 complex I plays essential roles in autophagosome formation. However, the regulatory mechanisms of yeast Vps34 complex I are still poorly understood. Here, we demonstrate that Atg1-dependent phosphorylation of Vps34 is required for robust autophagy activity in Saccharomyces cerevisiae. Following nitrogen starvation, Vps34 in complex I is selectively phosphorylated on multiple serine/threonine residues in its helical domain. This phosphorylation is important for full autophagy activation and cell survival. The absence of Atg1 or its kinase activity leads to complete loss of Vps34 phosphorylation in vivo, and Atg1 directly phosphorylates Vps34 in vitro, regardless of its complex association type. We also demonstrate that the localization of Vps34 complex I to the PAS provides a molecular basis for the complex I-specific phosphorylation of Vps34. This phosphorylation is required for the normal dynamics of Atg18 and Atg8 at the PAS. Together, our results reveal a novel regulatory mechanism of yeast Vps34 complex I and provide new insights into the Atg1-dependent dynamic regulation of the PAS.Abbreviations: ATG: autophagy-related; BARA: the repeated, autophagy-specific Co-IP: co-immunoprecipitation; GFP: green fluorescent protein; IP-MS: immunoprecipitation followed by tandem mass spectrometry; NTD: the N-terminal domain; PAS: phagophore assembly site; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns3K: phosphatidylinositol 3-kinase; SUR: structurally uncharacterized region; Vps34[KD]: Vps34D731N.

The trehalose-6-phosphate phosphatase Tps2 regulates ATG8 transcription and autophagy in Saccharomyces cerevisiae.

Macroautophagy/autophagy is an important catabolic process for maintaining cellular homeostasis by adapting to various stress conditions. Autophagy is mediated by a double-membrane autophagosome, which sequesters a portion of cytoplasmic components for delivery to the vacuole. Several autophagy-related (ATG) genes play crucial roles in autophagosome formation. The induction of ATG genes must be tightly regulated to maintain a proper autophagic activity, but their regulatory mechanisms are still largely unknown. Here, we report that the trehalose-6-phosphate phosphatase Tps2 functions as a positive regulator of autophagy in Saccharomyces cerevisiae. Cellular trehalose levels do not affect autophagy regulation by Tps2. Loss of Tps2 leads to impaired autophagic flux and reduced ATG8 expre/ssion under nitrogen starvation. In tps2Δ cells, Ume6 is predominantly dephosphorylated and represses ATG8 transcription by binding to its promoter region. Tps2 regulates nuclear translocation and activation of Rim15 kinase, a negative regulator of Ume6, by causing the dissociation of Rim15 from the 14-3-3 proteins Bmh1/2 under nitrogen starvation, suggesting that Rim15 mediates the function of Tps2 as a positive regulator of ATG8 induction. Furthermore, Tps2 plays a crucial role in the dephosphorylation of Ser1061 and Thr1075 residues of Rim15, which is important for controlling the dissociation of Rim15 from Bmh1/2 under nitrogen starvation. Together, our results reveal the role of Tps2 as a positive regulator of autophagy and provide new insight into the regulatory mechanisms of ATG gene expression.Abbreviations: ATG: autophagy-related; ChIP: chromatin immunoprecipitation; Co-IP: co-immunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; GFP: green fluorescent protein; PKA: protein kinase A; PtdIns3K: phosphatidylinositol 3-kinase; Rim15KI: kinase-inactive Rim15; Rim15-2A: Rim15S1061A,T1075A; TEM: transmission electron microscopy; TORC1: target of rapamycin complex 1.

Calculations of the binding affinities of protein-protein complexes with the fast multipole method.

In this paper, we used a coarse-grained model at the residue level to calculate the binding free energies of three protein-protein complexes. General formulations to calculate the electrostatic binding free energy and the van der Waals free energy are presented by solving linearized Poisson-Boltzmann equations using the boundary element method in combination with the fast multipole method. The residue level model with the fast multipole method allows us to efficiently investigate how the mutations on the active site of the protein-protein interface affect the changes in binding affinities of protein complexes. Good correlations between the calculated results and the experimental ones indicate that our model can capture the dominant contributions to the protein-protein interactions. At the same time, additional effects on protein binding due to atomic details are also discussed in the context of the limitations of such a coarse-grained model.

Aggregation of Chameleon Peptides: Implications of α-Helicity in Fibril Formation.

We investigate the relationship between the inherent secondary structure and aggregation propensity of peptides containing chameleon sequences (i.e., sequences that can adopt either α or ß structure depending on context) using a combination of replica exchange molecular dynamics simulations, ion-mobility mass spectrometry, circular dichroism, and transmission electron microscopy. We focus on an eight-residue long chameleon sequence that can adopt an α-helical structure in the context of the iron-binding protein from Bacillus anthracis (PDB id 1JIG ) and a ß-strand in the context of the baculovirus P35 protein (PDB id 1P35 ). We show that the isolated chameleon sequence is intrinsically disordered, interconverting between α-helical and ß-rich conformations. The inherent conformational plasticity of the sequence can be constrained by addition of flanking residues with a given secondary structure propensity. Intriguingly, we show that the chameleon sequence with helical flanking residues aggregates rapidly into fibrils, whereas the chameleon sequence with flanking residues that favor ß-conformations has weak aggregation propensity. This work sheds new insights into the possible role of α-helical intermediates in fibril formation.

Calculations of the second virial coefficients of protein solutions with an extended fast multipole method.

The osmotic second virial coefficients B(2) are directly related to the solubility of protein molecules in electrolyte solutions and can be useful to narrow down the search parameter space of protein crystallization conditions. Using a residue level model of protein-protein interaction in electrolyte solutions B(2) of bovine pancreatic trypsin inhibitor and lysozyme in various solution conditions such as salt concentration, pH and temperature are calculated using an extended fast multipole method in combination with the boundary element formulation. Overall, the calculated B(2) are well correlated with the experimental observations for various solution conditions. In combination with our previous work on the binding affinity calculations it is reasonable to expect that our residue level model can be used as a reliable model to describe protein-protein interaction in solutions.