Optimization of protein–protein docking for predicting Fc–protein interactions

The antibody crystallizable fragment (Fc) is recognized by effector proteins as part of the immune system. Pathogens produce proteins that bind Fc in order to subvert or evade the immune response. The structural characterization of the determinants of Fc–protein association is essential to improve our understanding of the immune system at the molecular level and to develop new therapeutic agents. Furthermore, Fc‐binding peptides and proteins are frequently used to purify therapeutic antibodies. Although several structures of Fc–protein complexes are available, numerous others have not yet been determined. Protein–protein docking could be used to investigate Fc–protein complexes; however, improved approaches are necessary to efficiently model such cases. In this study, a docking‐based structural bioinformatics approach is developed for predicting the structures of Fc–protein complexes. Based on the available set of X‐ray structures of Fc–protein complexes, three regions of the Fc, loosely corresponding to three turns within the structure, were defined as containing the essential features for protein recognition and used as restraints to filter the initial docking search. Rescoring the filtered poses with an optimal scoring strategy provided a success rate of approximately 80% of the test cases examined within the top ranked 20 poses, compared to approximately 20% by the initial unrestrained docking. The developed docking protocol provides a significant improvement over the initial unrestrained docking and will be valuable for predicting the structures of currently undetermined Fc–protein complexes, as well as in the design of peptides and proteins that target Fc.     

Iron–sulfur protein maturation in Helicobacter pylori: identifying a Nfu‐type cluster carrier protein and its iron–sulfur protein targets

Helicobacter pylori is anomalous among non nitrogen‐fixing bacteria in containing an incomplete NIF system for Fe–S cluster assembly comprising two essential proteins, NifS (cysteine desulfurase) and NifU (scaffold protein). Although nifU deletion strains cannot be obtained via the conventional gene replacement, a NifU‐depleted strain was constructed and shown to be more sensitive to oxidative stress compared to wild‐type (WT) strains. The hp1492 gene, encoding a putative Nfu‐type Fe–S cluster carrier protein, was disrupted in three different H. pylori strains, indicating that it is not essential. However, Δnfu strains have growth deficiency, are more sensitive to oxidative stress and are unable to colonize mouse stomachs. Moreover, Δnfu strains have lower aconitase activity but higher hydrogenase activity than the WT. Recombinant Nfu was found to bind either one [2Fe–2S] or [4Fe–4S] cluster/dimer, based on analytical, UV–visible absorption/CD and resonance Raman studies. A bacterial two‐hybrid system was used to ascertain interactions between Nfu, NifS, NifU and each of 36 putative Fe–S‐containing target proteins. Nfu, NifS and NifU were found to interact with 15, 6 and 29 putative Fe–S proteins respectively. The results indicate that Nfu, NifS and NifU play a major role in the biosynthesis and/or delivery of Fe–S clusters in H. pylori.     

Role of partial protein unfolding in alcohol‐induced protein aggregation

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease‐related as well as nondisease‐related proteins. Here we probed the biophysical mechanisms underlying alcohol‐induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site‐specific optical probes, two‐dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins. Proteins 2010. © Wiley‐Liss, Inc.     

Single-cell protein analysis by mass spectrometry

Human physiology and pathology arise from the coordinated interactions of diverse single cells. However, analyzing single cells has been limited by the low sensitivity and throughput of analytical methods. DNA sequencing has recently made such analysis feasible for nucleic acids but single-cell protein analysis remains limited. Mass spectrometry is the most powerful method for protein analysis, but its application to single cells faces three major challenges: efficiently delivering proteins/peptides to mass spectrometry detectors, identifying their sequences, and scaling the analysis to many thousands of single cells. These challenges have motivated corresponding solutions, including SCoPE design multiplexing and clean, automated, and miniaturized sample preparation. Synergistically applied, these solutions enable quantifying thousands of proteins across many single cells and establish a solid foundation for further advances. Building upon this foundation, the SCoPE concept will enable analyzing subcellular organelles and posttranslational modifications, while increases in multiplexing capabilities will increase the throughput and decrease cost.     

XRCC1 protein; Form and function

The human gene that encodes XRCC1 was cloned nearly thirty years ago but experimental analysis of this fascinating protein is still unveiling new insights into the DNA damage response. XRCC1 is a molecular scaffold protein that interacts with multiple enzymatic components of DNA single-strand break repair (SSBR) including DNA kinase, DNA phosphatase, DNA polymerase, DNA deadenylase, and DNA ligase activities that collectively are capable of accelerating the repair of a broad range of DNA single-strand breaks (SSBs). Arguably the most exciting aspect of XRCC1 function that has emerged in the last few years is its intimate relationship with PARP1 activity and critical role in preventing hereditary neurodegenerative disease. Here, I provide an update on our current understanding of XRCC1, and on the impact of hereditary mutations in this protein and its protein partners on human disease.     

The ups and downs of protein topology; rapid comparison of protein structure

Protein topology can be described at different levels. At the most fundamental level, it is a sequence of secondary structure elements (a "primary topology string"). Searching predicted primary topology strings against a library of strings from known protein structures is the basis of some protein fold recognition methods. Here a method known as TOPSCAN is presented for rapid comparison of protein structures. Rather than a simple two-letter alphabet (encoding strand and helix), more complex alphabets are used encoding direction, proximity, accessibility and length of secondary elements and loops in addition to secondary structure. Comparisons are made between the structural information content of primary topology strings and encodings which contain additional information ("secondary topology strings"). The algorithm is extremely fast, with a scan of a large domain against a library of more than 2000 secondary structure strings completing in approximately 30 s. Analysis of protein fold similarity using TOPSCAN at primary and secondary topology levels is presented.     

Jumonji domain‐containing protein 6 protein and its role in cancer

The jumonji domain‐containing protein 6 (JMJD6) is a Fe(II)‐ and 2‐oxoglutarate (2OG)‐dependent oxygenase that catalyses lysine hydroxylation and arginine demethylation of histone and non‐histone peptides. Recently, the intrinsic tyrosine kinase activity of JMJD6 has also been reported. The JMJD6 has been implicated in embryonic development, cellular proliferation and migration, self‐tolerance induction in the thymus, and adipocyte differentiation. Not surprisingly, abnormal expression of JMJD6 may contribute to the development of many diseases, such as neuropathic pain, foot‐and‐mouth disease, gestational diabetes mellitus, hepatitis C and various types of cancer. In the present review, we summarized the structure and functions of JMJD6, with particular emphasis on the role of JMJD6 in cancer progression.     

Protein <Emphasis Type="Italic">N</Emphasis>-glycosylation,protein folding,and protein quality control

Quality control of protein folding represents a fundamental cellular activity. Early steps of protein N-glycosylation involving the removal of three glucose and some specific mannose residues in the endoplasmic reticulum have been recognized as being of importance for protein quality control. Specific oligosaccharide structures resulting from the oligosaccharide processing may represent a glycocode promoting productive protein folding, whereas others may represent glyco-codes for routing not correctly folded proteins for dislocation from the endoplasmic reticulum to the cytosol and subsequent degradation. Although quality control of protein folding is essential for the proper functioning of cells, it is also the basis for protein folding disorders since the recognition and elimination of non-native conformers can result either in loss-of-function or pathological-gain-of-function. The machinery for protein folding control represents a prime example of an intricate interactome present in a single organelle, the endoplasmic reticulum. Here, current views of mechanisms for the recognition and retention leading to productive protein folding or the eventual elimination of misfolded glycoproteins in yeast and mammalian cells are reviewed.     

Rotaviral nonstructural protein 4 triggers dynamin‐related protein 1‐dependent mitochondrial fragmentation during infection

Dynamic equilibrium between mitochondrial fission and mitochondrial fusion serves as an important quality control system within cells ensuring cellular vitality and homeostasis. Viruses often target mitochondrial dynamics as a part of their obligatory cellular reprogramming. The present study was undertaken to assess the status and regulation of mitochondrial dynamics during rotavirus infection. Distinct fragmentation of mitochondrial syncytia was observed during late hours of RV (SA11, Wa, A5‐13) infection. RV nonstructural protein 4 (NSP4) was identified as the viral trigger for disrupted mitochondrial morphology. Severance of mitochondrial interconnections was found to be a dynamin‐related protein 1 (Drp1)‐dependent process resulting synergistically from augmented mitochondrial fission and attenuated mitochondrial fusion. Cyclin‐dependent kinase 1 was subsequently identified as the cellular kinase responsible for fission‐active Ser616 phosphorylation of Drp1. In addition to its positive role in mitochondrial fission, Drp1 also resulted in mitochondrial translocation of E3‐ubiquitin ligase Parkin leading to degradation of mitochondrial fusion protein Mitofusin 1. Interestingly, RV‐NSP4 was found to interact with and be involved in recruiting fission‐active pool of Serine 616 phosphoDrp1 (Ser616 pDrp1) to mitochondria independent of accessory adaptors Mitochondrial fission factor and Fission protein 1 (Fis1). Inhibition of either Drp1 or Ser616 pDrp1 resulted in significant decrease in RV‐NSP4‐induced intrinsic apoptotic pathway. Overall, this study underscores an efficient strategy utilised by RV to couple apoptosis to mitochondrial fission facilitating dissemination of viral progeny.