A receptor presentation hypothesis for T cell help that recruits autoreactive B cells

To uncover mechanisms that drive spontaneous expansions of autoreactive B cells in systemic lupus erythematosus, we analyzed somatic mutations in variable region genes expressed by a panel of (NZB x SWR)F(1) hybridomas representing a large, spontaneously arising clone with specificity for chromatin. A single mutation within the Jkappa intron that was shared by all members of the lineage indicated that the clone emanated from a single mutated precursor cell and led to the prediction that a somatic mutation producing a functionally decisive amino acid change in the coding region would also be universally shared. Upon cloning and sequencing the corresponding germline V(H) gene, we found that two replacement somatic mutations in FR1 and CDR2 were indeed shared by all seven clone members. Surprisingly, neither mutation influenced Ab binding to chromatin; however, one of them produced a nonconservative amino acid replacement in a mutationally "cold" region of FR1 and created an immunodominant epitope for class II MHC-restricted T cells. The epitope was restricted by IA(q) (SWR), and the SWR MHC locus is associated with systemic lupus erythematosus in (NZB x SWR)F(1) mice. These, and related findings, provoke the hypothesis that autoreactive B cells may be recruited by a "receptor presentation" mechanism involving cognate interactions between T cells and somatically generated V region peptides that are self-presented by B cells.     

Cotranslational Folding Increases GFP Folding Yield

Protein sequences evolved to fold in cells, including cotranslational folding of nascent polypeptide chains during their synthesis by the ribosome. The vectorial (N- to C-terminal) nature of cotranslational folding constrains the conformations of the nascent polypeptide chain in a manner not experienced by full-length chains diluted out of denaturant. We are still discovering to what extent these constraints affect later, posttranslational folding events. Here we directly address whether conformational constraints imposed by cotranslational folding affect the partitioning between productive folding to the native structure versus aggregation. We isolated polyribosomes from Escherichia coli cells expressing GFP, analyzed the nascent chain length distribution to determine the number of nascent chains that were long enough to fold to the native fluorescent structure, and calculated the folding yield for these nascent chains upon ribosome release versus the folding yield of an equivalent concentration of full-length, chemically denatured GFP polypeptide chains. We find that the yield of native fluorescent GFP is dramatically higher upon ribosome release of nascent chains versus dilution of full-length chains from denaturant. For kinetically trapped native structures such as GFP, folding correctly the first time, immediately after release from the ribosome, can lead to lifelong population of the native structure, as opposed to aggregation.     

Serine racemase: an unconventional enzyme for an unconventional transmitter

The discovery of large amounts of d-serine in the brain challenged the dogma that only l-amino acids are relevant for eukaryotes. The levels of d-serine in the brain are higher than many l-amino acids and account for as much as one-third of l-serine levels. Several studies in the last decades have demonstrated a role of d-serine as an endogenous agonist of N-methyl-d-aspartate receptors (NMDARs). d-Serine is required for NMDAR activity during normal neurotransmission as well as NMDAR overactivation that takes place in neurodegenerative conditions. Still, there are many unanswered questions about d-serine neurobiology, including regulation of its synthesis, release and metabolism. Here, we review the mechanisms of d-serine synthesis by serine racemase and discuss the lessons we can learn from serine racemase knockout mice, focusing on the roles attributed to d-serine and its cellular origin.     

GRASPing unconventional secretion

GRASP proteins associate with the Golgi apparatus and have been implicated in the stacking of Golgi cisternae, vesicle tethering, and mitotic progression, but their specific functions are unclear. In this issue, Kinseth et al. (2007) show unexpectedly that a GRASP homolog is required for an unconventional secretory pathway that bypasses the usual route for Golgi-dependent membrane traffic.     

蛋白质的折叠

重点介绍了蛋白质折叠的热力学控制学说和动力学控制学说,简单介绍了几种蛋白质折叠模型并分析了多肽链在体内进行快速折叠的原因。     

Functions of unconventional myosins

To date, fourteen classes of unconventional myosins have been identified. Recent reports have implicated a number of these myosins in organelle transport, and in the formation, maintenance and/or dynamics of actin-rich structures involved in a variety of cellular processes including endocytosis, cell migration, and sensory transduction. Characterizations of organelle dynamics in pigment cells and neurons have further defined the contributions made by unconventional myosins and microtubule motors to the transport and distribution of organelles. Several studies have provided evidence of complexes through which cooperative organelle transport may be coordinated. Finally, the myosin superfamily has been shown to contain at least one processive motor and one backwards motor.     

Folding properties of the hepatitis B core as a carrier protein for vaccination research

The hepatitis B core (HBc) protein has been used successfully in numerous experiments as a carrier for heterologous peptides. Folding and capsid formation of the chimeric proteins is not always achieved easily. In silico analyses were performed to provide further comprehension of the feasibility for predicting successful capsid formation. In contrast to previous work, we show that common in silico predictions do not ensure assembly into particles. We included new considerations regarding capsid formation of HBc fusion proteins. Not only the primary sequence and the length of the inserts seem important, also the rigidity, the distance between the N and the C-terminus and the presence of cysteines, which could form disulphide bonds, could influence proper capsid formation. Furthermore, new conformational insights were formulated when linkers were added to create extra flexibility of the chimeric particles. Different hypotheses were suggested to clarify the obtained results. To this extent, the addition of glycine-rich linkers could lower high rigidity of the insert, removal of the strain of the core protein or ease interaction between the HBc and the insert. Finally, we observed specific changes in capsid formation properties when longer linkers were used. These findings have not been reported before in this and other virus-like particle carriers. In this study, we also propose a new high-yield purification protocol for fusion proteins to be used in vaccination experiments with the carrier protein or in comparative studies of particulate or non-particulate HBc fusion proteins.     

Searching for life in the Universe: unconventional methods for an unconventional problem

The search for life, on and off our planet, can be done by conventional methods with which we are all familiar. These methods are sensitive and specific, and are often capable of detecting even single cells. However, if the search broadens to include life that may be different (even subtly different) in composition, the methods and even the approach must be altered. Here we discuss the development of what we call non-earthcentric life detection – detecting life with methods that could detect life no matter what its form or composition. To develop these methods, we simply ask, can we define life in terms of its general properties and particularly those that can be measured and quantified? Taking such an approach we can search for life using physics and chemistry to ask questions about structure, chemical composition, thermodynamics, and kinetics. Structural complexity can be searched for using computer algorithms that recognize complex structures. Once identified, these structures can be examined for a variety of chemical traits, including elemental composition, chirality, and complex chemistry. A second approach involves defining our environment in terms of energy sources (i.e., reductants), and oxidants (e.g. what is available to eat and breathe), and then looking for areas in which such phenomena are inexplicably out of chemical equilibrium. These disequilibria, when found, can then be examined in detail for the presence of the structural and chemical complexity that presumably characterizes any living systems. By this approach, we move the search for life to one that should facilitate the detection of any earthly life it encountered, as well as any non-conventional life forms that have structure, complex chemistry, and live via some form of redox chemistry. Electronic Publication     

Cotranslational Protein Folding

The review analyzes the research concerning the folding of proteins in the course of their synthesis on ribosomes. The experimental data obtained for various proteins using various methods give grounds for concluding that a nascent protein largely acquires its spatial structure while still attached to the ribosome, and final folding into the biologically active conformation takes place as soon as the completed protein is released therefrom. Cotranslational folding is characteristic of both bacterial and eukaryotic cells, and appears to be the universal and the most evolutionarily ancient mechanism.     

Chaperonin-mediated Protein Folding

We have been studying chaperonins these past twenty years through an initial discovery of an action in protein folding, analysis of structure, and elucidation of mechanism. Some of the highlights of these studies were presented recently upon sharing the honor of the 2013 Herbert Tabor Award with my early collaborator, Ulrich Hartl, at the annual meeting of the American Society for Biochemistry and Molecular Biology in Boston. Here, some of the major findings are recounted, particularly recognizing my collaborators, describing how I met them and how our great times together propelled our thinking and experiments.     

Folding of beta-hairpins

Identification of the factors governing the formation of beta-structure independently of the rest of the protein is important for understanding the folding process of protein into a unique native structure. It has been shown that some beta-hairpins can fold autonomously into native-like structures, either in aqueous solution or in the presence of an organic co-solvent. Our aim is to review recent theoretical and experimental studies of folding of beta-structures.