Advances in the study of luminescence probes for proteins

Spectral probes (or labels) have been widely used for the investigation and determination of proteins and have made considerable progress. Traditional luminescence probes include fluorescent derivatizing reagents, fluorescent probes and chemiluminescence probes which continue to develop. Of them, near infrared (NIR) fluorescent probes are especially suitable for the determination of biomolecules including proteins, so their development has been rapid. Novel luminescence probes (such as nanoparticle probes and molecular beacons) and resonance light scattering probes recently appeared in the literature. Preliminary results indicate that they possess great potential for ultrasensitive protein detection. This review summarizes recent developments of the above-mentioned probes for proteins and 195 references are cited.     

钙荧光探剂的研究及其在生命科学中的应用

钙荧光探剂测量活细胞胞浆游离Ｃａ２＋浓度的方法在钙研究中已成为一种越来越重要的技术。特别是由于新的一代荧光探剂的合成和激光共聚焦显微镜的发展,使其应用更加广泛。由于国内使用这种技术的实验室逐渐增多,本文将系统介绍钙荧光探剂的发展、测量原理和方法、新的常用钙荧光探剂的比较及其在生命科学中的应用。     

Recent advances in HDAC-targeted imaging probes for cancer detection

Histone Deacetylases (HDACs) are abnormally high expressed in various cancers and play a crucial role in regulating gene expression. While HDAC-targeted inhibitors have been rapidly developed and approved in the last twenty years, noninvasive monitoring and visualizing the expression levels of HDACs in tumor tissues might help to early diagnosis in cancer and predict the response to HDAC-targeted cancer therapy. In this review, we summarize the recent advancements in the development of HDAC-targeted probes and their applications in cancer imaging and image-guided surgery. We also discuss the design strategies, advantages and disadvantages of these probes. We hope that this review will provide guidance for the design of HDAC-targeted imaging probes and clinical applications in future.     

Activity-Based Imaging Probes of the Proteasome

Over the years, the proteasome has been extensively investigated due to its crucial roles in many important signaling pathways and its implications in diseases. Two proteasome inhibitors—bortezomib and carfilzomib—have received FDA approval for the treatment of multiple myeloma, thereby validating the proteasome as a chemotherapeutic target. As a result, further research efforts have been focused on dissecting the complex biology of the proteasome to gain the insight required for developing next-generation proteasome inhibitors. It is clear that chemical probes have made significant contributions to these efforts, mostly by functioning as inhibitors that selectively block the catalytic activity of proteasomes. Analogues of these inhibitors are now providing additional tools for visualization of catalytically active proteasome subunits, several of which allow real-time monitoring of proteasome activity in living cells as well as in in vivo settings. These imaging probes will provide powerful tools for assessing the efficacy of proteasome inhibitors in clinical settings. In this review, we will focus on the recent efforts towards developing imaging probes of proteasomes, including the latest developments in immunoproteasome-selective imaging probes.     

Phage display for detection of biological threat agents

The essential element of any immuno-based detector device is the probe that binds analyte and, as a part of the analytical platform, generates a measurable signal. The present review summarizes the state of the art in development of the probes for detection of the biological threat agents: toxins, bacteria, spores and viruses. Traditionally, the probes are antibodies, which are isolated from sera of immunized animals or culture media of hybridomas. However, the "natural" antibodies may have limited application in the new generation of real-time field detectors and monitoring systems, where stress-resistant and inexpensive long-livers are required. Phage display is a newcomer in the detection area, whose expertise is development of molecular probes for targeting of various biological structures. The probes can be selection from about billion clone libraries of recombinant phages expressing on their surface a vast variety of peptides and proteins, including antigen-binding fragments of antibodies. The selection procedure, like kind of affinity chromatography, allows separating of phage binders, which are propagated in Escherichia coli bacterial cells and purified using inexpensive technology. Although phage display traditionally is focused more on development of medical preparations and studying molecular recognition in biological systems, there are some examples of its successful use for detection, which are presented in the review. To be used as probes for detection, peptides and antibodies identified by phage display are usually chemically synthesized or produced in bacteria. Another interesting aspect is using of the selected phage itself as a probe in detector devices, like sort of substitute antibodies. This idea is illustrated in the review by "detection" of beta-galactosidase from E. coli with "landscape" phage displaying a dense array of peptide binders on the surface.     

Elucidation of enzyme mechanisms using fluorinated substrate analogues

A great variety of biological reactions that are physiologically important are catalyzed by enzymes. Understanding the reaction course of these enzyme-catalyzed transformations are of significant importance since the insights gained from these experiments may facilitate the design of methods to control or mimic their actions. A common strategy to study enzyme catalyses is to use fluorinated substrate analogues as mechanistic probes, since fluorine is an effective hydroxyl group mimic and can also be used to replace a hydrogen atom. Using fluorinated substrate probes have enabled researchers to obtain crucial information regarding the catalytic mechanism of enzymatic reactions. Many of these compounds are good enzyme inhibitors and have been developed into clinically useful chemotherapeutic agents. This review will discuss some examples of the use of fluorine containing compounds as mechanistic probes/enzyme inhibitors, many of which are selected from our own work.     

Development of functional gene microarrays for microbial community analysis

Functional gene arrays (FGAs) are a special type of microarrays containing probes for key genes involved in microbial functional processes, such as biogeochemical cycling of carbon, nitrogen, sulfur, phosphorus and metals, virulence and antibiotic resistance, biodegradation of environmental contaminants, and stress responses. FGAs have been demonstrated to be a specific, sensitive, and quantitative tool for rapid analysis of microbial communities from different habitats, such as waters, soils, extreme environments, bioreactors, and human microbiomes. In this review, we first summarize currently reported FGAs, and then focus on the FGA development. We will also discuss several key issues of FGA technology as well as challenges and directions in future FGA development.     

Protein-based tumor molecular imaging probes

Molecular imaging is an emerging discipline which plays critical roles in diagnosis and therapeutics. It visualizes and quantifies markers that are aberrantly expressed during the disease origin and development. Protein molecules remain to be one major class of imaging probes, and the option has been widely diversified due to the recent advances in protein engineering techniques. Antibodies are part of the immunosystem which interact with target antigens with high specificity and affinity. They have long been investigated as imaging probes and were coupled with imaging motifs such as radioisotopes for that purpose. However, the relatively large size of antibodies leads to a half-life that is too long for common imaging purposes. Besides, it may also cause a poor tissue penetration rate and thus compromise some medical applications. It is under this context that various engineered protein probes, essentially antibody fragments, protein scaffolds, and natural ligands have been developed. Compared to intact antibodies, they possess more compact size, shorter clearance time, and better tumor penetration. One major challenge of using protein probes in molecular imaging is the affected biological activity resulted from random labeling. Site-specific modification, however, allows conjugation happening in a stoichiometric fashion with little perturbation of protein activity. The present review will discuss protein-based probes with focus on their application and related site-specific conjugation strategies in tumor imaging.     

Detection of ubiquitin-proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development

Background: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin-proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics.     

Improved methods for targeting epigenetic reader domains of acetylated and methylated lysine

Responsible for interpreting histone post-translational modifications, epigenetic reader proteins have emerged as novel therapeutic targets for a wide range of diseases. Chemical probes have been critical in enabling target validation studies and have led to translational advances in cancer and inflammation-related pathologies. Here, we present the most recently reported probes of reader proteins that recognize acylated and methylated lysine. We will discuss challenges associated with achieving potent antagonism of reader domains and review ongoing efforts to overcome these hurdles, focusing on targeting strategies including the use of peptidomimetic ligands, allosteric modulators, and protein degraders.     

The synthesis and compositional analysis of phosphopeptides

Protein phosphorylation plays a critical role in the regulation of cell growth and differentiation, There is considerable interest, therefore, in the facile synthesis of peptides that possess selectively phosphorylated residues for use as molecular probes in mechanistic studies of the biological consequences of phosphorylation. This work will review the various synthetic protocols used in the generation of phosphopeptides and will discuss their characterization by amino acid compositional analysis.     

Proteomics meets microbiology: technical advances in the global mapping of protein expression and function

The availability of complete genome sequences for a large number of pathogenic organisms has opened the door for large-scale proteomic studies to dissect both protein expression/regulation and function. This review highlights key proteomic methods including two-dimensional gel electrophoresis, reference mapping, protein expression profiling and recent advances in gel-free separation techniques that have made a significant impact on the resolution of complex proteomes. In addition, we highlight recent developments in the field of chemical proteomics, a branch of proteomics aimed at functionally profiling a proteome. These techniques include the development of activity-based probes and activity-based protein profiling methods as well as the use of synthetic small molecule libraries to screen for pharmacological tools to perturb basic biological processes. This review will focus on the applications of these technologies to the field of microbiology.     

LightUp probes in clinical diagnostics

The LightUp Probe technology has now matured and reached the phase where it has been implemented in commercial reagent kits, i.e. the ReSSQ product line. Several properties of the LightUp probes make them particularly suitable for clinical settings. For instance, extraordinary shelf life and a chemical stability that allows convenient fridge storage. The origin of the higher stability of LightUp probe kits compared to others, based on alternative probe technologies, is partly the relatively good stability of cyanine dyes but also the resistance towards nucleases and proteases of the synthetic DNA analogue peptide nucleic acid that is used as the sequence recognizing element in LightUp probes. It is clear from recent trends in the PCR amplification hardware technology that the instrumentation is becoming more flexible and less adapted for dedicated probe chemistries. This will pave the way for increased standardization in the field of DNA diagnostics and the development of cross-platform assays. In the present review the LightUp technology will briefly be presented and discussed. The utility of the technology will be illustrated by examples from cytomegalovirus quantification and monitoring of the viral load of the SARS Coronavirus. An example of cancer diagnostics by detection of altered gene expression patterns will also be shown.     

Photocrosslinking probes for capture of carbohydrate interactions

Glycan-mediated interactions are essential in many biological processes and regulate a wide variety of cellular functions. However, characterizing these interactions is difficult because glycan biosynthesis is not template driven and because carbohydrate recognition events are usually of low affinity and transient. Photocrosslinking carbohydrate probes can form a covalent bond with molecules in close proximity on UV irradiation and are capable of capturing interactions between glycans and glycan-binding proteins in situ. Because of these advantages, multiple photocrosslinking carbohydrate probes have been designed and applied to study the biological functions of glycans. This review will discuss recent advances in the development of novel photocrosslinking functional groups and the design of photocrosslinking probes to detect interactions mediated by glycolipids, peptidoglycan, and multivalent carbohydrate ligands. These probes have demonstrated the potential to address some of the major challenges in the study of glycan-mediated interactions in both model systems and in more complex biological settings.     

Chemical-biological studies of subcellular organization in bacteria

The subcellular organization of biological molecules is a critical determinant of many bacterial processes, including growth, replication of the genome, and division, yet the details of many mechanisms that control intracellular organization remain unknown. Decoding this information will impact the field of bacterial physiology and can provide insight into eukaryotic biology, including related processes in mitochondria and chloroplasts. Small molecule probes provide unique advantages in studying these mechanisms and manipulating the organization of biomolecules in live bacterial cells. In this review, we describe small molecules that are available for investigating subcellular organization in bacteria, specifically targeting FtsZ, MreB, peptidoglycan, and lipid bilayers. We discuss how these probes have been used to study microbiological questions and conclude by providing suggestions about important areas in which chemical-biological approaches will have a revolutionary impact on the study of bacterial physiology.     

Mitochondrial membrane potential probes and the proton gradient: a practical usage guide

Fluorescent probes for monitoring mitochondrial membrane potential are frequently used for assessing mitochondrial function, particularly in the context of cell fate determination in biological and biomedical research. However, valid interpretation of results obtained with such probes requires careful consideration of numerous controls, as well as possible effects of non-protonic charges on dye behavior. In this context, we provide an overview of some of the important technical considerations, controls, and parallel complementary assays that can be employed to help ensure appropriate interpretation of results, thus providing a practical usage guide for monitoring mitochondrial membrane potentials with cationic probes. In total, this review will help illustrate both the strengths and potential pitfalls of common mitochondrial membrane potential dyes, and highlight best-usage approaches for their efficacious application in life sciences research.     

On the molecular mechanisms of mitotic kinase activation

During mitosis, human cells exhibit a peak of protein phosphorylation that alters the behaviour of a significant proportion of proteins, driving a dramatic transformation in the cell''s shape, intracellular structures and biochemistry. These mitotic phosphorylation events are catalysed by several families of protein kinases, including Auroras, Cdks, Plks, Neks, Bubs, Haspin and Mps1/TTK. The catalytic activities of these kinases are activated by phosphorylation and through protein–protein interactions. In this review, we summarize the current state of knowledge of the structural basis of mitotic kinase activation mechanisms. This review aims to provide a clear and comprehensive primer on these mechanisms to a broad community of researchers, bringing together the common themes, and highlighting specific differences. Along the way, we have uncovered some features of these proteins that have previously gone unreported, and identified unexplored questions for future work. The dysregulation of mitotic kinases is associated with proliferative disorders such as cancer, and structural biology will continue to play a critical role in the development of chemical probes used to interrogate disease biology and applied to the treatment of patients.     

Two-photon probes for biomedical applications

Two-photon microscopy (TPM), which uses two photons of lower energy as the excitation source, is a vital tool in biology and clinical science, due to its capacity to image deep inside intact tissues for a long period of time. To make TPM a more versatile tool in biomedical research, we have developed a variety of two-photon probes for specific applications. In this mini review, we will briefly discuss two-photon probes for lipid rafts, lysosomes, mitochondria, and pH, and their biomedical applications. [BMB Reports 2013; 46(4): 188-194]