Favorite Protein Research Articles

Let’s get biophysical – How to get your favorite protein’s digits

In these days of information overload and high-throughput analysis, it is easy to lose focus on the study of individual proteins. It is our conjecture that such investigations are still crucially important and offer uniquely penetrative insights. We thus present a discussion of biophysical methods to allow readers to get to know their protein of interest better. Although this perspective is not written with the expert in mind, we hope that for interdisciplinary scientists, or researchers who do not routinely perform biophysical analyses, the content will be helpful and inspiring.

The Structural Dynamics, Complexity of Interactions, and Functions in Cancer of Multi-SAM Containing Proteins.

SAM domains are crucial mediators of diverse interactions, including those important for tumorigenesis or metastasis of cancers, and thus SAM domains can be attractive targets for developing cancer therapies. This review aims to explore the literature, especially on the recent findings of the structural dynamics, regulation, and functions of SAM domains in proteins containing more than one SAM (multi-SAM containing proteins, MSCPs). The topics here include how intrinsic disorder of some SAMs and an additional SAM domain in MSCPs increase the complexity of their interactions and oligomerization arrangements. Many similarities exist among these MSCPs, including their effects on cancer cell adhesion, migration, and metastasis. In addition, they are all involved in some types of receptor-mediated signaling and neurology-related functions or diseases, although the specific receptors and functions vary. This review also provides a simple outline of methods for studying protein domains, which may help non-structural biologists to reach out and build new collaborations to study their favorite protein domains/regions. Overall, this review aims to provide representative examples of various scenarios that may provide clues to better understand the roles of SAM domains and MSCPs in cancer in general.

Antibody There? Mapping the Human Proteome with Mathias Uhlén

Antibody There? Mapping the Human Proteome with Mathias Uhlén

Abstract PL05-03: What if all your favorite proteins are druggable

Abstract Many proteins that play key roles in cancer are considered difficult drug targets, if not entirely undruggable, due to an apparent lack of pockets where small molecules can bind with the affinity and specificity required for a drug. ‘Cryptic’ pockets that are absent in known structures of proteins but form due to protein dynamics could alleviate this problem. However, it has been hard to assess or exploit the opportunities that cryptic pockets present due to the inherent difficulty in identifying such pockets. Here, I will discuss progress from my lab on combining biophysical experiments, computer simulations, and machine learning to identify and target cryptic pockets. In one case study, we have used computer simulations deployed on the Folding@home distributed computing environment to predict a cryptic pocket that can allosterically control a protein-RNA interaction, and then experimentally confirmed our predictions. Based on data from this study and others in our lab, we have trained a machine learning algorithm to predict where cryptic pockets are likely to form from single protein structures, enabling us to assess how prevalent cryptic pockets are on a large scale. Citation Format: Gregory R. Bowman. What if all your favorite proteins are druggable. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr PL05-03.

The Meaning of an End: N-Terminal Acetyltransferase NAA50 Controls Plant Growth and Stress Responses.

At least 80% of eukaryotic proteins are estimated to undergo N-terminal acetylation (NTA), making it likely that your favorite protein is also regulated by NTA ([Linster and Wirtz, 2018][1]). This transfer of an acetyl group to the N terminus can modulate a protein’s interaction partners, i.e.

Scientists unveil new DNA-encoded library

With a simple package in the mail, scientists can start studying how their favorite protein interacts with millions of small molecules. This is what researchers from the Novartis Institutes for Biomedical Research and the Broad Institute of MIT and Harvard said they hope will happen as they announced their new open-source DNA-encoded library (DEL) at the American Chemical Society national meeting in San Diego last week. DELs are large pools of small molecules, each attached to a ribbon of DNA with a unique sequence of base pairs. Researchers can look for molecules in the DEL that bind to the protein, sequence the DNA tags to find the binding molecules’ identities, and then synthesize them for future studies. The Novartis-Broad DEL is different from other open-source DELs because of the 3 million compounds it contains—many have unusual structures and well-defined stereochemistries, said Liam Hudson, a postdoc at Novartis and Broad. Many

Inferring the Evolutionary History of Your Favorite Protein: A Guide for Molecular Biologists.

Comparative genomics has proven a fruitful approach to acquire many functional and evolutionary insights into core cellular processes. Here it is argued that in order to perform accurate and interesting comparative genomics, one first and foremost has to be able to recognize, postulate, and revise different evolutionary scenarios. After all, these studies lack a simple protocol, due to different proteins having different evolutionary dynamics and demanding different approaches. The authors here discuss this challenge from a practical (what are the observations?) and conceptual (how do these indicate a specific evolutionary scenario?) viewpoint, with the aim to guide investigators who want to analyze the evolution of their protein(s) of interest. By sharing how the authors draft, test, and update such a scenario and how it directs their investigations, the authors hope to illuminate how to execute molecular evolution studies and how to interpret them. Also see the video abstract here https://youtu.be/VCt3l2pbdbQ.

Convert your favorite protein modeling program into a mutation predictor: "MODICT".

BackgroundPredict whether a mutation is deleterious based on the custom 3D model of a protein.ResultsWe have developed modict, a mutation prediction tool which is based on per residue rmsd (root mean square deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin (REN), beta-tubulin (TUBB2B), biotinidase (BTD), sphingomyelin phosphodiesterase-1 (SMPD1), phenylalanine hydroxylase (PAH) and medium chain Acyl-Coa dehydrogenase (ACADM). Moreover, modict scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The modict perl package and the manual can be downloaded from https://github.com/IbrahimTanyalcin/MODICT.ConclusionsWe show here that modict is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by polyphen and sift.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1286-0) contains supplementary material, which is available to authorized users.

A robust method to screen detergents for membrane protein stabilization, revisited

This report is a follow up of our previous paper (Lund, Orlowski, de Foresta, Champeil, le Maire and Møller (1989), J Biol Chem 264:4907–4915) showing that solubilization in detergent of a membrane protein may interfere with its long-term stability, and proposing a protocol to reveal the kinetics of such irreversible inactivation. We here clarify the fact that when various detergents are tested for their effects, special attention has of course to be paid to their critical micelle concentration. We also investigate the effects of a few more detergents, some of which have been recently advertised in the literature, and emphasize the role of lipids together with detergents. Among these detergents, lauryl maltose neopentyl glycol (LMNG) exerts a remarkable ability, even higher than that of β-dodecylmaltoside (DDM), to protect our test enzyme, the paradigmatic P-type ATPase SERCA1a from sarcoplasmic reticulum. Performing such experiments for one's favourite protein probably remains useful in pre-screening assays testing various detergents.

Forgotten fluids of the reproductive tract

Forgotten fluids of the reproductive tract

Detection of Protein Posttranslational Modifications from Whole-Cell Extracts in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is an ideal model organism as numerous cellular mechanisms are conserved in mammalian cells. This includes posttranslational modifications (PTMs) such as ubiquitination, sumoylation, and phosphorylation. For ubiquitination, target proteins are readily modified via a cascade reaction, which can result in various forms of ubiquitination known to be associated with numerous cellular mechanisms. Therefore it becomes imperative for researchers to detect PTMs of their favorite proteins in order to determine how the target proteins function and are regulated. However, detection of ubiquitination in vivo, as well as some other PTMs, has proven challenging for researchers due to the presence of deconjugating enzymes in the cell. This chapter describes a step-by-step protocol on how to preserve and subsequently detect PTMs of your favorite protein from budding yeast S. cerevisiae whole-cell extracts.

BACs to the future: new genetic models for cardiovascular discovery

One of the great challenges of modern biology is to contextualize highly detailed molecular discoveries in order to prioritize the most medically relevant events. The response to heart damage, for example, likely involves tens of thousands of cells of multiple lineages, each undergoing complex signalling cascades that trigger other equally complex molecular interactions including cell migration, lineage induction, secretion, fibrosis, and revascularization. In determining the relevance of discrete molecular events within these broader contexts, we often infer their involvement from experiments in isolated cells under somewhat artificial conditions. How do we know that these exquisitely specific events occur under realistic clinical conditions? If so, to what extent? At what point in the process do they occur (acute damage, repair, etc.)? And perhaps most importantly, are they critical to the process? In short, how do we answer as early as possible the ‘so what’ question with respect to our favourite protein–protein interaction, transcriptional induction, or other molecular event, and thereby avoid spending years studying a potential epiphenomenon? One approach is to do …

Selective Autophagy: Talking with the UPS

Far from now are the days when investigators raced to identify the proteolytic system responsible for the degradation of their favorite protein. Nowadays, it is well accepted that a given protein can be degraded by different systems depending on factors such as cell type, cellular conditions, or functionality of each proteolytic pathway. The realization of this sharing of substrates among pathways has also helped to unveil deeper levels of communication among the different proteolytic systems. Thus, cells often respond to blockage of one degradative mechanism by upregulating any of the other available pathways. In addition, effectors and regulators of one proteolytic system can be degraded by a different proteolytic pathway that exerts, in this way, a regulatory function. In this mini review, we describe the different levels of cross-talk among autophagic pathways and the ubiquitin/proteasome system. We also provide examples of how this proteolytic communication is used for compensatory purposes in different pathological conditions and discuss the possible therapeutic potential of targeting the modulators of the cross-talk among proteolytic pathways.

The mFruit Collection of Monomeric Fluorescent Proteins

Featured Article: Shaner NC, Campbell RE, Steinbach PA, Giepmans BN, Palmer AE, Tsien RY. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 2004:22;1567–72.2 Fluorescent proteins, starting with the green fluorescent protein (GFP)3 from Aequorea victoria, have revolutionized our ability to noninvasively study living systems (1). Practically every cell biologist has used fluorescent proteins to tag a favorite protein and watch its dynamic localization in living cells. For almost 10 years after the gene encoding GFP was cloned, researchers were limited to using only about half of the visible spectrum with these probes and were able to image only 2 different colors simultaneously, at best. In 1999, Mikhail Matz and colleagues at the Russian Academy of Science discovered the first known red fluorescent protein, in Discosoma sp. coral; that protein would become known as DsRed (2). Hopes were high that the wavelength barrier had been broken, and many researchers jumped at the chance to try this new color. Unfortunately, it soon …

Single molecules made simple

A single-molecule pull-down method provides a simple way for biologists to examine their favorite protein at the single-molecule level.

Sensitive protein microarray synergistically amplified by polymer brush-enhanced immobilizations of both probe and reporter

Great challenge remains to continuously improve sensitivity of protein microarrays for broad applications. A copolymer brush is in situ synthesized on both substrate and silica nanoparticle (SNP) surface to efficiently immobilize probe and reporter protein respectively for synergistic amplification of protein microarray signals. As a demonstration, sandwich immunoassay for a cancer biomarker carcinoembryonic antigen (CEA) detection is performed on microarray platform, showing a limit of detection (LOD) of 10pg/ml and dynamic range of 10pg/ml to 100ng/ml. Two orders improvement of LOD is achieved in comparison to the small crosslinker-activated substrate. The improved sensitivity is attributed to not only the high immobilization amount of both probe and reporter but also the favorite protein binding orientations offered by the flexible brushes. This work provides a universal approach to inexpensively and significantly improve protein microarray sensitivity.

Arabidopsis 14-3-3 Proteins: Fascinating and Less Fascinating Aspects

14-3-3 Dimers are well known to interact with diverse target proteins throughout eukaryotes. Most notably, association of 14-3-3s commonly requires phosphorylation of a serine or threonine residue within a specific sequence motif of the client protein. Studies with a focus on individual target proteins have unequivocally demonstrated 14-3-3s to be the crucial factors modifying the client’s activity state upon phosphorylation and, thus, finishing the job initiated by a kinase. In this respect, a recent in-depth analysis of the rice transcription factor FLOWERING LOCUS D1 (OsFD1) revealed 14-3-3s to be essential players in floral induction. Such fascinating discoveries, however, can often be ascribed to the random identification of 14-3-3 as an interaction partner of the favorite protein. In contrast, our understanding of 14-3-3 function in higher organisms is frustratingly limited, mainly due to an overwhelming spectrum of putative targets in combination with the existence of a multigene 14-3-3 family. In this review we will discuss our current understanding of the function of plant 14-3-3 proteins, taking into account recent surveys of the Arabidopsis 14-3-3 interactome.

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

HotSpot Wizard: a web server for identification of hot spots in protein engineering

HotSpot Wizard is a web server for automatic identification of ‘hot spots’ for engineering of substrate specificity, activity or enantioselectivity of enzymes and for annotation of protein structures. The web server implements the protein engineering protocol, which targets evolutionarily variable amino acid positions located in the active site or lining the access tunnels. The ‘hot spots’ for mutagenesis are selected through the integration of structural, functional and evolutionary information obtained from: (i) the databases RCSB PDB, UniProt, PDBSWS, Catalytic Site Atlas and nr NCBI and (ii) the tools CASTp, CAVER, BLAST, CD-HIT, MUSCLE and Rate4Site. The protein structure and e-mail address are the only obligatory inputs for the calculation. In the output, HotSpot Wizard lists annotated residues ordered by estimated mutability. The results of the analysis are mapped on the enzyme structure and visualized in the web browser using Jmol. The HotSpot Wizard server should be useful for protein engineers interested in exploring the structure of their favourite protein and for the design of mutations in site-directed mutagenesis and focused directed evolution experiments. HotSpot Wizard is available at http://loschmidt.chemi.muni.cz/hotspotwizard/.