Chapter 3: Small Molecules and Disease

“Big” molecules such as proteins and genes still continue to capture the imagination of most biologists, biochemists and bioinformaticians. “Small” molecules, on the other hand, are the molecules that most biologists, biochemists and bioinformaticians prefer to ignore. However, it is becoming increasingly apparent that small molecules such as amino acids, lipids and sugars play a far more important role in all aspects of disease etiology and disease treatment than we realized. This particular chapter focuses on an emerging field of bioinformatics called “chemical bioinformatics” – a discipline that has evolved to help address the blended chemical and molecular biological needs of toxicogenomics, pharmacogenomics, metabolomics and systems biology. In the following pages we will cover several topics related to chemical bioinformatics. First, a brief overview of some of the most important or useful chemical bioinformatic resources will be given. Second, a more detailed overview will be given on those particular resources that allow researchers to connect small molecules to diseases. This section will focus on describing a number of recently developed databases or knowledgebases that explicitly relate small molecules – either as the treatment, symptom or cause – to disease. Finally a short discussion will be provided on newly emerging software tools that exploit these databases as a means to discover new biomarkers or even new treatments for disease.

What to Learn in This Chapter

• The meaning of chemical bioinformatics
• Strengths and limitations of existing chemical bioinformatic databases
• Using databases to learn about the cause and treatment of diseases
• The Small Molecule Pathway Database (SMPDB)
• The Human Metabolome Database (HMDB)
• DrugBank
• The Toxin and Toxin-Target Database (T3DB)
• PolySearch and Metabolite Set Enrichment Analysis

This article is part of the “Translational Bioinformatics” collection for PLOS Computational Biology.

     

Cobalt carbonyl complexes as probes for alkyne-tagged lipids

Monitoring lipid distribution and metabolism in cells and biological fluids poses many challenges because of the many molecular species and metabolic pathways that exist. This study describes the synthesis and study of molecules that contain an alkyne functional group as surrogates for natural lipids in cultured cells. Thus, hexadec-15-ynoic and hexadec-7-ynoic acids were readily incorporated into RAW 264.7 cells, principally as phosphocholine esters; the alkyne was used as a “tag” that could be transformed to a stable dicobalt-hexacarbonyl complex; and the complex could then be detected by HPLC/MS or HPLC/UV_349nm. The 349 nm absorbance of the cobalt complexes was used to provide qualitative and quantitative information about the distribution and cellular concentrations of the alkyne lipids. The alkyne group could also be used as an affinity tag for the lipids by a catch-and-release strategy on phosphine-coated silica beads. Lipid extracts were enriched in the tagged lipids in this way, making the approach of potential utility to study lipid transformations in cell culture. Both terminal alkynes and internal alkynes were used in this affinity “pull-down” strategy. This method facilitates measuring lipid species that might otherwise fall below limits of detection.     

The Potential Regulatory Mechanisms of miR-196a in Huntington’s Disease through Bioinformatic Analyses

High throughput screening is a powerful tool to identify the potential candidate molecules involved during disease progression. However, analysis of complicated data is one of the most challenging steps on the way to obtaining useful results from this approach. Previously, we showed that a specific miRNA, miR-196a, could ameliorate the pathological phenotypes of Huntington’s disease (HD) in different models, and performed high throughput screening by using the striatum of transgenic mice. In this study, we further tried to identify the potential regulatory mechanisms using different bioinformatic tools, including Database for Annotation, Visualization and Integrated Discovery (DAVID), Molecular Signatures Database (MSigDB), TargetScan and MetaCore. The results showed that miR-196a dominantly altered “ABC transporters”, “RIG-I-like receptor signaling pathway”, immune system”, “adaptive immune system”,“tissue remodeling and wound repair” and “cytoskeleton remodeling”. In addition, miR-196a also changed the expression of several well-defined pathways of HD, such as apoptosis and cell adhesion. Since these analyses showed the regulatory pathways are highly related to the modification of the cytoskeleton, we further confirmed that miR-196a could enhance the neurite outgrowth in neuroblastoma cells, suggesting miR-196a might provide beneficial functions through the alteration of cytoskeleton structures. Since impairment of the cytoskeleton has been reported in several neuronal diseases, this study will provide not only the potential working mechanisms of miR-196a but also insights for therapeutic strategies for use with different neuronal diseases.     

Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer''s Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer's yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.     

Action of Deflocculating Agents on Saccharomyces cerevisiae Class III Brewer's Yeast

Earlier work with a fluorescent aid indicated that flocculent brewer''s yeast may have more surface lipids than nonflocculent types. Organic solvents were checked against flocculent Gilliland yeasts. It was found that those reagents which affect “free” lipids had no dispersive action, and those which remove “bound” fats had a powerful dispersive action against such yeasts. There was no indication that such an action could be correlated with other physical properties of the solvents. The uranyl ion is known for its ability to complex with phospholipids, and it was found to have a powerful dispersive action on Gilliland yeasts. Its effect was compared with that of glucose in its dispersion of yeast flocs, and possible cell “sites” were suggested. This, along with other work, suggests the possibility that lipids are directly or indirectly involved in yeast flocculation.     

The Coomassie chronicles: past,present and future perspectives in polyacrylamide gel staining

In recent years, MALDI imaging mass spectrometry (MALDI-IMS) has developed as a promising tool to investigate the spatial distribution of biomolecules in intact tissue specimens. Ion densities of various molecules can be displayed as heat maps while preserving anatomical structures. In this short review, an overview of different biomolecules that can be analyzed by MALDI-IMS is given. Many reviews have covered imaging of lipids, small metabolites, whole proteins and enzymatically digested proteins in the past. However, little is known about imaging of endogenous peptides, for example, in the rat brain, and this will therefore be highlighted in this review. Furthermore, sample preparation of frozen or formalin-fixed, paraffin-embedded (FFPE) tissue is crucial for imaging experiments. Therefore, some aspects of sample preparation will be addressed, including washing and desalting, the choice of MALDI matrix and its deposition. Apart from mapping endogenous peptides, their reliable identification in situ still remains challenging and will be discussed as well.     

MALDI imaging mass spectrometry: molecular snapshots of biochemical systems

Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is emerging as a powerful tool for investigating the distribution of molecules within biological systems through the direct analysis of thin tissue sections. Unique among imaging methods, MALDI-IMS can determine the distribution of hundreds of unknown compounds in a single measurement. We discuss the current state of the art of MALDI-IMS along with some recent applications and technological developments that illustrate not only its current capabilities but also the future potential of the technique to provide a better understanding of the underlying molecular mechanisms of biological processes.     

Phospholipid Flip-Flop and the Distribution of Surface Charges in Excitable Membranes

There is now good evidence that most of the lipids in a biological membrane are arranged in the form of a bilayer. Charged lipids in the membrane of an excitable cell are subject to a significant driving force, the gradient of the intramembrane potential, which will tend to redistribute the lipids between the two halves of the bilayer by a “phospholipid flip-flop” mechanism. We have calculated, by combining the Boltzmann relation from statistics and the Gouy equation from the theory of the diffuse double layer, the steady-state distribution of charged lipids in the bilayer. This distribution is completely determined, within the framework of the model, by three experimentally accessible variables; the percentage of charged lipid in the bilayer as a whole, the resting potential and the ionic strength. The known values for the percentage of anionic phospholipids in squid axons (10-15%), the membrane potential (50-100 mV) and ionic strength (0.5 M) imply that the charge density and double layer potential at the outer surface of the nerve will be substantially greater than the charge density and double layer potential at the inner surface, in agreement with the best available evidence from physiological measurements.     

Logic Gate Operation by DNA Translocation through Biological Nanopores

Logical operations using biological molecules, such as DNA computing or programmable diagnosis using DNA, have recently received attention. Challenges remain with respect to the development of such systems, including label-free output detection and the rapidity of operation. Here, we propose integration of biological nanopores with DNA molecules for development of a logical operating system. We configured outputs “1” and “0” as single-stranded DNA (ssDNA) that is or is not translocated through a nanopore; unlabeled DNA was detected electrically. A negative-AND (NAND) operation was successfully conducted within approximately 10 min, which is rapid compared with previous studies using unlabeled DNA. In addition, this operation was executed in a four-droplet network. DNA molecules and associated information were transferred among droplets via biological nanopores. This system would facilitate linking of molecules and electronic interfaces. Thus, it could be applied to molecular robotics, genetic engineering, and even medical diagnosis and treatment.     

An Imputation Approach for Oligonucleotide Microarrays

Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as “bright spots”, “dark clouds”, and “shadowy circles”, etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.     

Lipids in cell biology: how can we understand them better?

Lipids are a major class of biological molecules and play many key roles in different processes. The diversity of lipids is on the same order of magnitude as that of proteins: cells express tens of thousands of different lipids and hundreds of proteins to regulate their metabolism and transport. Despite their clear importance and essential functions, lipids have not been as well studied as proteins. We discuss here some of the reasons why it has been challenging to study lipids and outline technological developments that are allowing us to begin lifting lipids out of their “Cinderella” status. We focus on recent advances in lipid identification, visualization, and investigation of their biophysics and perturbations and suggest that the field has sufficiently advanced to encourage broader investigation into these intriguing molecules.Lipids are fundamental building blocks of all cells and play many important and varied roles. They are key components of the plasma membrane and other cellular compartments, including the nuclear membrane, the endoplasmic reticulum, the Golgi apparatus, and trafficking vesicles such as endosomes and lysosomes. The lipid composition of different organelles, cell types, and ultimately tissues can vary substantially, suggesting that different lipids are required for different functions (Saghatelian et al., 2006 ; Klose et al., 2013 ). Mammalian cells express tens of thousands of different lipid species and use hundreds of proteins to synthesize, metabolize, and transport them. Although the complexity and diversity of lipids approach those of proteins, we have a much poorer understanding of their functions, making lipids in many ways the “Cinderellas” of cell biology. We discuss here recent advances, and challenges, in investigating how these molecules contribute to the many biological processes in which they participate.Like proteins, lipids can have structural (e.g., by stabilizing different membrane curvatures) or signaling roles. Posttranslational lipidation of proteins (e.g., palmitoylation or farnesylation) and carbohydrate-linked lipids (glycolipids) are also important examples of cellular lipid pools. It is clear that higher-order organization is key to most lipid functions, and they are believed to assemble into signaling platforms that contain both lipids and proteins (Kusumi et al., 2012 ). Some lipid microdomains are termed lipid rafts, and much ongoing effort is being focused on defining their parameters (Simons and Sampaio, 2011 ; Suzuki et al., 2012 ; Klotzsch and Schutz, 2013 ). Because of this focus on lipid rafts, we have a better understanding of the properties of proposed raft lipids (e.g., sphingomyelins and cholesterol) than of many other lipid species. Much of what we know about lipids has come from studying synthetic membranes with specific lipid compositions. Although model membranes usually consist of very few (<10) lipid species, they have been useful for understanding the biophysical properties of lipids. At the other end of the spectrum, lipids have been implicated in various diseases, with cholesterol metabolism being a prominent example. We are now getting to a point at which we can address the roles of lipids in the middle of the spectrum—in cells.     

A Molecular Structural Basis for the Excitation Properties of Axons

A structural model is suggested for axon membranes consisting of a double layer of lipid and phospholipid molecules in which the polar ends of certain phospholipids change their orientation and combining properties under the influence of an electric field. The phosphate groups act as ion exchange “gates” for the control of ion flow through the membrane. Expressions are developed for the calculation of membrane current components as functions of time, potential, and ionic environment. Approximate solutions show fairly good agreement with existing experimental data in a number of different respects such as steady-state current-voltage relations, the effect of calcium on steady-state current, potassium tracer flux ratios, initial current and rate of change of current, and the dependence of the time constants of current change on membrane potential.     

Imaging mass spectrometry for lipidomics

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a powerful tool that enables the simultaneous detection and identification of biomolecules in analytes. MALDI-imaging mass spectrometry (MALDI-IMS) is a two-dimensional MALDI-MS technique used to visualize the spatial distribution of biomolecules without extraction, purification, separation, or labeling of biological samples. This technique can reveal the distribution of hundreds of ion signals in a single measurement and also helps in understanding the cellular profile of the biological system. MALDI-IMS has already revealed the characteristic distribution of several kinds of lipids in various tissues. The versatility of MALDI-IMS has opened a new frontier in several fields, especially in lipidomics. In this review, we describe the methodology and applications of MALDI-IMS to biological samples.     

Weighting of Criteria for Disease Prioritization Using Conjoint Analysis and Based on Health Professional and Student Opinion

Disease prioritization exercises have been used by several organizations to inform surveillance and control measures. Though most methodologies for disease prioritization are based on expert opinion, it is becoming more common to include different stakeholders in the prioritization exercise. This study was performed to compare the weighting of disease criteria, and the consequent prioritization of zoonoses, by both health professionals and students in Switzerland using a Conjoint Analysis questionnaire. The health professionals comprised public health and food safety experts, cantonal physicians and cantonal veterinarians, while the student group comprised first-year veterinary and agronomy students. Eight criteria were selected for this prioritization based on expert elicitation and literature review. These criteria, described on a 3-tiered scale, were evaluated through a choice-based Conjoint Analysis questionnaire with 25 choice tasks. Questionnaire results were analyzed to obtain importance scores (for each criterion) and mean utility values (for each criterion level), and the latter were then used to rank 16 zoonoses. While the most important criterion for both groups was “Severity of the disease in humans”, the second ranked criteria by the health professionals and students were “Economy” and “Treatment in humans”, respectively. Regarding the criterion “Control and Prevention”, health professionals tended to prioritize a disease when the control and preventive measures were described to be 95% effective, while students prioritized a disease if there were almost no control and preventive measures available. Bovine Spongiform Encephalopathy was the top-ranked disease by both groups. Health professionals and students agreed on the weighting of certain criteria such as “Severity” and “Treatment of disease in humans”, but disagreed on others such as “Economy” or “Control and Prevention”. Nonetheless, the overall disease ranking lists were similar, and these may be taken into consideration when making future decisions regarding resource allocation for disease control and prevention in Switzerland.     

Choline-releasing glycerophosphodiesterase EDI3 links the tumor metabolome to signaling network activities

Recently, EDI3 was identified as a key factor for choline metabolism that controls tumor cell migration and is associated with metastasis in endometrial carcinomas. EDI3 cleaves glycerophosphocholine (GPC) to form choline and glycerol-3-phosphate (G3P). Choline is then further metabolized to phosphatidylcholine (PtdC), the major lipid in membranes and a key player in membrane-mediated cell signaling. The second product, G3P, is a precursor molecule for several lipids with central roles in signaling, for example lysophosphatidic acid (LPA), phosphatidic acid (PA) and diacylglycerol (DAG). LPA activates intracellular signaling pathways by binding to specific LPA receptors, including membrane-bound G protein-coupled receptors and the intracellular nuclear receptor, PPARγ. Conversely, PA and DAG mediate signaling by acting as lipid anchors that bind and activate several signaling proteins. For example, binding of GTPases and PKC to PA and DAG, respectively, increases the activation of signaling networks, mediating processes such as migration, adhesion, proliferation or anti-apoptosis—all relevant for tumor development. We present a concept by which EDI3 either directly generates signaling molecules or provides “membrane anchors” for downstream signaling factors. As a result, EDI3 links choline metabolism to signaling activities resulting in a more malignant phenotype.     

Comparison of Predictive Value of Cardiometabolic Indices for Subclinical Atherosclerosis in Chinese Adults

Objectives

Metabolic disturbances are well-known risk factors for atherosclerosis, but it remains unclear which cardiometabolic components are the predominant determinants. This study aimed to compare and identify the key determinants of carotid atherosclerosis in asymptomatic middle-aged and elderly Chinese.

Methods

A community-based cross-sectional study including 3,162 apparently healthy residents aged 37–75 years was performed from July 2008 to June 2010 in Guangzhou, China. Carotid artery intima-media thickness (IMT) was assessed by B-mode ultrasound, and increased IMT was defined as IMT>1.00 mm. Obesity indices, blood pressure, fasting blood lipids, glucose and uric acid levels were determined. Principal components factor analysis was used to extract common factors underlying 11 metabolic factors.

Results

Four common factors, defined as “adiposity,” “blood lipids,” “triglycerides/uric acid (TG/UA)” (in men) or “triglycerides/uric acid/glucose (TG/UA/Glu)” (in women), and “blood pressure,” were retained for both sexes. After adjustment for potential covariates, the “adiposity” factor showed the strongest positive association with increased IMT in men. Comparing the extreme quartiles, ORs (95% CI) of increased IMT were 4.64 (2.04–10.59) at the CCA and 2.37 (1.54–3.64) at the BIF), followed by “blood pressure”, the corresponding OR (95% CI) was 2.85 (1.37–5.90) at the CCA. Whereas, the four common factors showed comparable and weak relationship with increased IMTs, the ORs for quartile 4 vs. quartile 1 varied from 0.89 to 3.59 in women.

Conclusions

Among the metabolic factors, “adiposity” and “blood pressure” play predominant roles in the presence of carotid atherosclerosis in men, but no key factor is identified in women.     

Prioritizing Disease Candidate Proteins in Cardiomyopathy-Specific Protein-Protein Interaction Networks Based on “Guilt by Association” Analysis

The cardiomyopathies are a group of heart muscle diseases which can be inherited (familial). Identifying potential disease-related proteins is important to understand mechanisms of cardiomyopathies. Experimental identification of cardiomyophthies is costly and labour-intensive. In contrast, bioinformatics approach has a competitive advantage over experimental method. Based on “guilt by association” analysis, we prioritized candidate proteins involving in human cardiomyopathies. We first built weighted human cardiomyopathy-specific protein-protein interaction networks for three subtypes of cardiomyopathies using the known disease proteins from Online Mendelian Inheritance in Man as seeds. We then developed a method in prioritizing disease candidate proteins to rank candidate proteins in the network based on “guilt by association” analysis. It was found that most candidate proteins with high scores shared disease-related pathways with disease seed proteins. These top ranked candidate proteins were related with the corresponding disease subtypes, and were potential disease-related proteins. Cross-validation and comparison with other methods indicated that our approach could be used for the identification of potentially novel disease proteins, which may provide insights into cardiomyopathy-related mechanisms in a more comprehensive and integrated way.     

Recent insights into atherosclerotic plaque cell autophagy

Cardiovascular and cerebrovascular diseases, such as coronary heart disease and stroke, caused by atherosclerosis have become the “number one killer”, seriously endangering human health in developing and developed countries. Atherosclerosis mainly occurs in large and medium-sized arteries and involves intimal thickening, accumulation of foam cells, and formation of atheromatous plaques. Autophagy is a cellular catabolic process that has evolved to defend cells from the turnover of intracellular molecules. Autophagy is thought to play an important role in the development of plaques. This review focuses on studies on autophagy in cells involved in the formation of atherosclerotic plaques, such as monocytes, macrophages, endothelial cells, dendritic cells, and vascular smooth muscle cells, indicating that autophagy plays an important role in plaque development. We mainly discuss the roles of autophagy in these cells in maintaining the stability of atherosclerotic plaques, providing a reference for the next steps to unravel the mechanisms of atherogenesis.     

Multivalent protein–protein interactions are pivotal regulators of eukaryotic Hsp70 complexes

Heat shock protein 70 (Hsp70) is a molecular chaperone and central regulator of protein homeostasis (proteostasis). Paramount to this role is Hsp70’s binding to client proteins and co-chaperones to produce distinct complexes, such that understanding the protein–protein interactions (PPIs) of Hsp70 is foundational to describing its function and dysfunction in disease. Mounting evidence suggests that these PPIs include both “canonical” interactions, which are universally conserved, and “non-canonical” (or “secondary”) contacts that seem to have emerged in eukaryotes. These two categories of interactions involve discrete binding surfaces, such that some clients and co-chaperones engage Hsp70 with at least two points of contact. While the contributions of canonical interactions to chaperone function are becoming increasingly clear, it can be challenging to deconvolute the roles of secondary interactions. Here, we review what is known about non-canonical contacts and highlight examples where their contributions have been parsed, giving rise to a model in which Hsp70’s secondary contacts are not simply sites of additional avidity but are necessary and sufficient to impart unique functions. From this perspective, we propose that further exploration of non-canonical contacts will generate important insights into the evolution of Hsp70 systems and inspire new approaches for developing small molecules that tune Hsp70-mediated proteostasis.