APOBEC mutagenesis and selection for NFE2L2 contribute to the origin of lung squamous-cell carcinoma.

Lung squamous-cell carcinoma originates as a consequence of oncogenic molecular variants arising from diverse mutagenic processes such as tobacco, defective homologous recombination, aging, and cytidine deamination by APOBEC proteins. Only some of the many variants generated by these processes actually contribute to tumorigenesis. Therefore, molecular investigation of mutagenic processes such as cytidine deamination by APOBEC should also determine whether the mutations produced by these processes contribute substantially to the growth and survival of cancer. Here, we determine the processes that gave rise to mutations of 681 lung squamous-cell carcinomas, and quantify the probability that each mutation was the product of each process. We then calculate the contribution of each mutation to increases in cellular proliferation and survival. We performed in vitro experiments to determine cytidine deamination activity of APOBEC3B against oligonucleotides corresponding with genomic sequences that give rise to variants of high cancer effect size. The largest APOBEC-related cancer effects are attributable to mutations in PIK3CA and NFE2L2. We demonstrate that APOBEC effectively deaminates NFE2L2 at the locations that confer high cancer effect. Overall, we demonstrate that APOBEC activity can lead to mutations in NFE2L2 that have large contributions to cancer cell growth and survival, and that NFE2L2 is an attractive potential target for therapeutic intervention.

N-Doped Carbon Quantum Dot (NCQD)-Deposited Carbon Capsules for Synergistic Fluorescence Imaging and Photothermal Therapy of Oral Cancer.

Use of nanomaterials blessed with both therapeutic and diagnostic properties is a proficient strategy in the treatment of cancer in its early stage. In this context, our paper reports the synthesis of uniform size N-rich mesoporous carbon nanospheres of size 65-70 nm from pyrrole and aniline precursors using Triton-X as a structure-directing agent. Transmission electron microscopy reveals that these carbons spheres contain void spaces in which ultrasmall nitrogen-doped quantum dots (NCQD) are captured within the matrix. These mesoporous hollow NCQD captured carbon spheres (NCQD-HCS) show fluorescence quantum yield up to 14.6% under λex = 340 nm. Interestingly, samples calcined at >800 °C clearly absorb in the wavelength range 700-1000 nm and shows light-to-heat conversion efficiency up to 52%. In vitro experiments in human oral cancer cells (FaDu) show that NCQD-HCS are internalized by the cells and induce a substantial thermal ablation effect in FaDu cells when exposed under a 980 nm near-infrared laser.

Ion Mobility Mass Spectrometry Uncovers the Impact of the Patterning of Oppositely Charged Residues on the Conformational Distributions of Intrinsically Disordered Proteins.

The global dimensions and amplitudes of conformational fluctuations of intrinsically disordered proteins are governed, in part, by the linear segregation versus clustering of oppositely charged residues within the primary sequence. Ion mobility-mass spectrometry (IM-MS) affords unique advantages for probing the conformational consequences of the linear patterning of oppositely charged residues because it measures and separates proteins electrosprayed from solution on the basis of charge and shape. Here, we use IM-MS to measure the conformational consequences of charge patterning on the C-terminal intrinsically disordered region (p27 IDR) of the cell cycle inhibitory protein p27Kip1. We report the range of charge states and accompanying collisional cross section distributions for wild-type p27 IDR and two variants with identical amino acid compositions, κ14 and κ56, distinguished by the extent of linear mixing versus segregation of oppositely charged residues. Wild-type p27 IDR (κ31) and κ14, where the oppositely charged residues are more evenly distributed, exhibit a broad distribution of charge states. This is concordant with high degrees of conformational heterogeneity in solution. By contrast, κ56 with linear segregation of oppositely charged residues leads to limited conformational heterogeneity and a narrow distribution of charged states. Gas-phase molecular dynamics simulations demonstrate that the interplay between chain solvation and intrachain interactions (self-solvation) leads to conformational distributions that are modulated by salt concentration, with the wild-type sequence showing the most sensitivity to changes in salt concentration. These results suggest that the charge patterning within the wild-type p27 IDR may be optimized to sample both highly solvated and self-solvated conformational states.

Magnetic Mesoporous Silica Gated with Doped Carbon Dot for Site-Specific Drug Delivery, Fluorescence, and MR Imaging.

Construction of a theranostic agent which integrates multiple modalities with different functions into one entity is challenging from a molecular design and synthesis perspective. In this context, the present paper reports the fabrication of a novel type of multifunctional hybrid nanoparticle composed of magnetic gadolinium oxide-iron oxide core, mesoporous silica shell gated with boronic acid functionalized highly luminescent carbon quantum dot (BNSCQD). The porous silica shell acts as an excellent reservoir for anticancer drug 5-fluorouracil, whereas the BNSCQD cap impressively controls the drug transport under simulated intracellular environment. Furthermore, recognition and fluorescence turn on response of BNSCQD toward cell surface glycan sialyl Lewisa (SLa) enables targeted drug release and excellent fluorescence imaging of SLa overexpressed HePG2 cancer cells. The r1 and r2 relaxivities of the material are found to be 10 and 165 mM-1 s-1 which is comparable to commercially available magnetic resonance imaging contrast agents. Benefiting from the combined advantages of dual stimuli-responsive drug release, excellent optical imaging, and MR imaging, this novel construct can be a promising theranostic material.

A High-Throughput Mutational Scan of an Intrinsically Disordered Acidic Transcriptional Activation Domain.

Transcriptional activation domains are essential for gene regulation, but their intrinsic disorder and low primary sequence conservation have made it difficult to identify the amino acid composition features that underlie their activity. Here, we describe a rational mutagenesis scheme that deconvolves the function of four activation domain sequence features-acidity, hydrophobicity, intrinsic disorder, and short linear motifs-by quantifying the activity of thousands of variants in vivo and simulating their conformational ensembles using an all-atom Monte Carlo approach. Our results with a canonical activation domain from the Saccharomyces cerevisiae transcription factor Gcn4 reconcile existing observations into a unified model of its function: the intrinsic disorder and acidic residues keep two hydrophobic motifs from driving collapse. Instead, the most-active variants keep their aromatic residues exposed to the solvent. Our results illustrate how the function of intrinsically disordered proteins can be revealed by high-throughput rational mutagenesis.

Control of transcriptional activity by design of charge patterning in the intrinsically disordered RAM region of the Notch receptor.

Intrinsically disordered regions (IDRs) play important roles in proteins that regulate gene expression. A prominent example is the intracellular domain of the Notch receptor (NICD), which regulates the transcription of Notch-responsive genes. The NICD sequence includes an intrinsically disordered RAM region and a conserved ankyrin (ANK) domain. The 111-residue RAM region mediates bivalent interactions of NICD with the transcription factor CSL. Although the sequence of RAM is poorly conserved, the linear patterning of oppositely charged residues shows minimal variation. The conformational properties of polyampholytic IDRs are governed as much by linear charge patterning as by overall charge content. Here, we used sequence design to assess how changing the charge patterning within RAM affects its conformational properties, the affinity of NICD to CSL, and Notch transcriptional activity. Increased segregation of oppositely charged residues leads to linear decreases in the global dimensions of RAM and decreases the affinity of a construct including a C-terminal ANK domain (RAMANK) for CSL. Increasing charge segregation from WT RAM sharply decreases transcriptional activation for all permutants. Activation also decreases for some, but not all, permutants with low charge segregation, although there is considerable variation. Our results suggest that the RAM linker is more than a passive tether, contributing local and/or long-range sequence features that modulate interactions within NICD and with downstream components of the Notch pathway. We propose that sequence features within IDRs have evolved to ensure an optimal balance of sequence-encoded conformational properties, interaction strengths, and cellular activities.

CIDER: Resources to Analyze Sequence-Ensemble Relationships of Intrinsically Disordered Proteins.

Intrinsically disordered proteins and regions (IDPs) represent a large class of proteins that are defined by conformational heterogeneity and lack of persistent tertiary/secondary structure. IDPs play important roles in a range of biological functions, and their dysregulation is central to numerous diseases, including neurodegeneration and cancer. The conformational ensembles of IDPs are encoded by their amino acid sequences. Here, we present two computational tools that are designed to enable rapid and high-throughput analyses of a wide range of physicochemical properties encoded by IDP sequences. The first, CIDER, is a user-friendly webserver that enables rapid analysis of IDP sequences. The second, localCIDER, is a high-performance software package that enables a wide range of analyses relevant to IDP sequences. In addition to introducing the two packages, we demonstrate the utility of these resources using examples where sequence analysis offers biophysical insights.

Cryptic sequence features within the disordered protein p27Kip1 regulate cell cycle signaling.

Peptide motifs embedded within intrinsically disordered regions (IDRs) of proteins are often the sites of posttranslational modifications that control cell-signaling pathways. How do IDR sequences modulate the functionalities of motifs? We answer this question using the polyampholytic C-terminal IDR of the cell cycle inhibitory protein p27(Kip1) (p27). Phosphorylation of Thr-187 (T187) within the p27 IDR controls entry into S phase of the cell division cycle. Additionally, the conformational properties of polyampholytic sequences are predicted to be influenced by the linear patterning of oppositely charged residues. Therefore, we designed sequence variants of the p27 IDR to alter charge patterning outside the primary substrate motif containing T187. Computer simulations and biophysical measurements confirm predictions regarding the impact of charge patterning on the global dimensions of IDRs. Through functional studies, we uncover cryptic sequence features within the p27 IDR that influence the efficiency of T187 phosphorylation. Specifically, we find a positive correlation between T187 phosphorylation efficiency and the weighted net charge per residue of an auxiliary motif. We also find that accumulation of positive charges within the auxiliary motif can diminish the efficiency of T187 phosphorylation because this increases the likelihood of long-range intra-IDR interactions that involve both the primary and auxiliary motifs and inhibit their contributions to function. Importantly, our findings suggest that the cryptic sequence features of the WT p27 IDR negatively regulate T187 phosphorylation signaling. Our approaches provide a generalizable strategy for uncovering the influence of sequence contexts on the functionalities of primary motifs in other IDRs.

Highly Hydrophilic Luminescent Magnetic Mesoporous Carbon Nanospheres for Controlled Release of Anticancer Drug and Multimodal Imaging.

Judicious combination of fluorescence and magnetic properties along with ample drug loading capacity and control release property remains a key challenge in the design of nanotheranostic agents. This paper reports the synthesis of highly hydrophilic optically traceable mesoporous carbon nanospheres which can sustain payloads of the anticancer drug doxorubicin and T2 contrast agent such as cobalt ferrite nanoparticles. The luminescent magnetic hybrid system has been prepared on a mesoporous silica template using a resorcinol-formaldehyde precursor. The mesoporous matrix shows controlled release of the aromatic drug doxorubicin due to disruption of supramolecular π-π interaction at acidic pH. The particles show MR contrast behavior by affecting the proton relaxation with transverse relaxivity (r2) 380 mM(-1) S(-1). The multicolored emission and upconversion luminescence property of our sample are advantageous in bioimaging. In vitro cell experiments shows that the hybrid nanoparticles are endocyted by the tumor cells through passive targeting. The pH-responsive release of doxorubicin presents chemotherapeutic inhibition of cell growth through induction of apoptosis.

Fuzzy regions in an intrinsically disordered protein impair protein-protein interactions.

Despite the partial disorder-to-order transition that intrinsically disordered proteins often undergo upon binding to their partners, a considerable amount of residual disorder may be retained in the bound form, resulting in a fuzzy complex. Fuzzy regions flanking molecular recognition elements may enable partner fishing through non-specific, transient contacts, thereby facilitating binding, but may also disfavor binding through various mechanisms. So far, few computational or experimental studies have addressed the effect of fuzzy appendages on partner recognition by intrinsically disordered proteins. In order to shed light onto this issue, we used the interaction between the intrinsically disordered C-terminal domain of the measles virus (MeV) nucleoprotein (NTAIL ) and the X domain (XD) of the viral phosphoprotein as model system. After binding to XD, the N-terminal region of NTAIL remains conspicuously disordered, with α-helical folding taking place only within a short molecular recognition element. To study the effect of the N-terminal fuzzy region on NTAIL /XD binding, we generated N-terminal truncation variants of NTAIL , and assessed their binding abilities towards XD. The results revealed that binding increases with shortening of the N-terminal fuzzy region, with this also being observed with hsp70 (another MeV NTAIL binding partner), and for the homologous NTAIL /XD pairs from the Nipah and Hendra viruses. Finally, similar results were obtained when the MeV NTAIL fuzzy region was replaced with a highly dissimilar artificial disordered sequence, supporting a sequence-independent inhibitory effect of the fuzzy region.

Relating sequence encoded information to form and function of intrinsically disordered proteins.

Intrinsically disordered proteins (IDPs) showcase the importance of conformational plasticity and heterogeneity in protein function. We summarize recent advances that connect information encoded in IDP sequences to their conformational properties and functions. We focus on insights obtained through a combination of atomistic simulations and biophysical measurements that are synthesized into a coherent framework using polymer physics theories.

A quantitative measure for protein conformational heterogeneity.

Conformational heterogeneity is a defining characteristic of proteins. Intrinsically disordered proteins (IDPs) and denatured state ensembles are extreme manifestations of this heterogeneity. Inferences regarding globule versus coil formation can be drawn from analysis of polymeric properties such as average size, shape, and density fluctuations. Here we introduce a new parameter to quantify the degree of conformational heterogeneity within an ensemble to complement polymeric descriptors. The design of this parameter is guided by the need to distinguish between systems that couple their unfolding-folding transitions with coil-to-globule transitions and those systems that undergo coil-to-globule transitions with no evidence of acquiring a homogeneous ensemble of conformations upon collapse. The approach is as follows: Each conformation in an ensemble is converted into a conformational vector where the elements are inter-residue distances. Similarity between pairs of conformations is quantified using the projection between the corresponding conformational vectors. An ensemble of conformations yields a distribution of pairwise projections, which is converted into a distribution of pairwise conformational dissimilarities. The first moment of this dissimilarity distribution is normalized against the first moment of the distribution obtained by comparing conformations from the ensemble of interest to conformations drawn from a Flory random coil model. The latter sets an upper bound on conformational heterogeneity thus ensuring that the proposed measure for intra-ensemble heterogeneity is properly calibrated and can be used to compare ensembles for different sequences and across different temperatures. The new measure of conformational heterogeneity will be useful in quantitative studies of coupled folding and binding of IDPs and in de novo sequence design efforts that are geared toward controlling the degree of heterogeneity in unbound forms of IDPs.

Conformations of intrinsically disordered proteins are influenced by linear sequence distributions of oppositely charged residues.

The functions of intrinsically disordered proteins (IDPs) are governed by relationships between information encoded in their amino acid sequences and the ensembles of conformations that they sample as autonomous units. Most IDPs are polyampholytes, with sequences that include both positively and negatively charged residues. Accordingly, we focus here on the sequence-ensemble relationships of polyampholytic IDPs. The fraction of charged residues discriminates between weak and strong polyampholytes. Using atomistic simulations, we show that weak polyampholytes form globules, whereas the conformational preferences of strong polyampholytes are determined by a combination of fraction of charged residues values and the linear sequence distributions of oppositely charged residues. We quantify the latter using a patterning parameter κ that lies between zero and one. The value of κ is low for well-mixed sequences, and in these sequences, intrachain electrostatic repulsions and attractions are counterbalanced, leading to the unmasking of preferences for conformations that resemble either self-avoiding random walks or generic Flory random coils. Segregation of oppositely charged residues within linear sequences leads to high κ-values and preferences for hairpin-like conformations caused by long-range electrostatic attractions induced by conformational fluctuations. We propose a scaling theory to explain the sequence-encoded conformational properties of strong polyampholytes. We show that naturally occurring strong polyampholytes have low κ-values, and this feature implies a selection for random coil ensembles. The design of sequences with different κ-values demonstrably alters the conformational preferences of polyampholytic IDPs, and this ability could become a useful tool for enabling direct inquiries into connections between sequence-ensemble relationships and functions of IDPs.

How is functional specificity achieved through disordered regions of proteins?

N-type inactivation of potassium channels is controlled by cytosolic loops that are intrinsically disordered. Recent experiments have shown that the mechanism of N-type inactivation through disordered regions can be stereospecific and vary depending on the channel type. Variations in mechanism occur despite shared coarse grain features such as the length and amino acid compositions of the cytosolic disordered regions. We have adapted a phenomenological model designed to explain how specificity in molecular recognition is achieved through disordered regions. We propose that the channel-specific observations for N-type inactivation represent distinct mechanistic choices for achieving function through conformational selection versus induced fit. It follows that the dominant mechanism for binding and specificity can be modulated through subtle changes in the amino acid sequences of disordered regions, which is interesting given that specificity in function is realized in the absence of autonomous folding.

Unmasking functional motifs within disordered regions of proteins.

Eukaryotic proteins often possess long stretches that fail to adopt well-defined, three-dimensional structures. These intrinsically disordered regions are associated with cell signaling through the enrichment of hub proteins of networks and as targets for posttranslational modifications. Although disordered regions are readily identified because of their distinct sequence characteristics, it is difficult to predict the functions associated with these regions. This is because disordered regions often house short (two- to five-residue) linear motifs that mediate intermolecular interactions. Predicting their function requires the ability to identify the functionally relevant motifs. If one assumes that functional motifs are highly conserved as compared to background sequence contexts, then a suitable comparative genomics approach proves to be powerful in unmasking functional motifs that are part of disordered regions. This approach has successfully identified known functional motifs and predicted a set of new motifs that might yield important insights regarding previously unknown functionalities for disordered regions. Given knowledge of highly conserved motifs, one can assess whether the rapidly changing sequence contexts are actuators of the functionalities of short linear motifs within disordered regions. This should have important implications for engineering and targeting hub proteins in signaling networks.

N-terminal segments modulate the α-helical propensities of the intrinsically disordered basic regions of bZIP proteins.

Basic region leucine zippers (bZIPs) are modular transcription factors that play key roles in eukaryotic gene regulation. The basic regions of bZIPs (bZIP-bRs) are necessary and sufficient for DNA binding and specificity. Bioinformatic predictions and spectroscopic studies suggest that unbound monomeric bZIP-bRs are uniformly disordered as isolated domains. Here, we test this assumption through a comparative characterization of conformational ensembles for 15 different bZIP-bRs using a combination of atomistic simulations and circular dichroism measurements. We find that bZIP-bRs have quantifiable preferences for α-helical conformations in their unbound monomeric forms. This helicity varies from one bZIP-bR to another despite a significant sequence similarity of the DNA binding motifs (DBMs). Our analysis reveals that intramolecular interactions between DBMs and eight-residue segments directly N-terminal to DBMs are the primary modulators of bZIP-bR helicities. We test the accuracy of this inference by designing chimeras of bZIP-bRs to have either increased or decreased overall helicities. Our results yield quantitative insights regarding the relationship between sequence and the degree of intrinsic disorder within bZIP-bRs, and might have general implications for other intrinsically disordered proteins. Understanding how natural sequence variations lead to modulation of disorder is likely to be important for understanding the evolution of specificity in molecular recognition through intrinsically disordered regions (IDRs).

Coupling between motor proteins determines dynamic behaviors of motor protein assemblies.

Transport of intracellular cargos by multiple microtubule motor proteins is believed to be a common and significant phenomenon in vivo, yet signatures of the microscopic dynamics of multiple motor systems are only now beginning to be resolved. Understanding these mechanisms largely depends on determining how grouping motors affect their association with microtubules and stepping rates, and hence, cargo run lengths and velocities. We examined this problem using a discrete state transition rate model of collective transport. This model accounts for the structural and mechanical properties in binding/unbinding and stepping transitions between distinct microtubule-bound configurations of a multiple motor system. In agreement with previous experiments that examine the dynamics of two coupled kinesin-1 motors, the energetic costs associated with deformations of mechanical linkages within a multiple motor assembly are found to reduce the system's overall microtubule affinity, producing attenuated mean cargo run lengths compared to cases where motors are assumed to function independently. With our present treatment, this attenuation largely stems from reductions in the microtubule binding rate and occurs even when mechanical coupling between motors is weak. Thus, our model suggests that, at least for a variety of kinesin-dependent transport processes, the net 'gains' obtained by grouping motors together may be smaller than previously expected.

Facilitated search of proteins on DNA: correlations are important.

A starting point of many biological processes is protein binding to specific regions on DNA. Although typical concentrations of DNA-binding proteins are low, and target sites are typically buried among huge number of non-specific sites, the search process is frequently achieved at a remarkably fast rate. For some proteins it has been confirmed that association rates might be even larger than the maximal allowed three-dimensional diffusion rates. The current theoretical view of this phenomenon is based on the idea of lowering dimensionality, i.e., the overall search process is viewed as a combination of uncorrelated three-dimensional excursions in the solution and one-dimensional hoppings on DNA. However, some predictions of this theoretical picture contradict recent single-molecule measurements of protein diffusion processes. An alternative theoretical approach points out the importance of correlations during the search process that appear due to non-specific interactions between protein and DNA molecules. To test different theoretical ideas we performed extensive lattice Monte Carlo computer simulations of the facilitated diffusion. Our results revealed that correlations are important, and the acceleration in the search could only be achieved at some intermediate non-specific binding energies and protein concentrations. Physico-chemical aspects and the origins of these correlations are discussed.