T cell receptor antagonism interferes with MHC clustering and integrin patterning during immunological synapse formation

T cell activation by nonself peptide-major histocompatibility complex (MHC) antigenic complexes can be blocked by particular sequence variants in a process termed T cell receptor antagonism. The inhibition mechanism is not understood, although such variants are encountered in viral infections and may aid immune evasion. Here, we study the effect of antagonist peptides on immunological synapse formation by T cells. This cellular communication process features early integrin engagement and T cell motility arrest, referred to as the "stop signal." We find that synapses formed on membranes presenting antagonist-agonist complexes display reduced MHC density, which leads to reduced T cell proliferation that is not overcome by the costimulatory ligands CD48 and B7-1. Most T cells fail to arrest and crawl slowly with a dense ICAM-1 crescent at the leading edge. Similar aberrant patterns of LFA-1/ICAM-1 engagement in live T-B couples correlate with reduced calcium flux and IL-2 secretion. Hence, antagonist peptides selectively disable MHC clustering and the stop signal, whereas LFA-1 valency up-regulation occurs normally.     

Subtleties in control by metabolic channelling and enzyme organization

Because of its importance to cell function, the free-energy metabolism of the living cell is subtly and homeostatically controlled. Metabolic control analysis enables a quantitative determination of what controls the relevant fluxes. However, the original metabolic control analysis was developed for idealized metabolic systems, which were assumed to lack enzyme-enzyme association and direct metabolite transfer between enzymes (channelling). We here review the recently developed molecular control analysis, which makes it possible to study non-ideal (channelled, organized) systems quantitatively in terms of what controls the fluxes, concentrations, and transit times. We show that in real, non-ideal pathways, the central control laws, such as the summation theorem for flux control, are richer than in ideal systems: the sum of the control of the enzymes participating in a non-ideal pathway may well exceed one (the number expected in the ideal pathways), but may also drop to values below one. Precise expressions indicate how total control is determined by non-ideal phenomena such as ternary complex formation (two enzymes, one metabolite), and enzyme sequestration. The bacterial phosphotransferase system (PTS), which catalyses the uptake and concomitant phosphorylation of glucose (and also regulates catabolite repression) is analyzed as an experimental example of a non-ideal pathway. Here, the phosphoryl group is channelled between enzymes, which could increase the sum of the enzyme control coefficients to two, whereas the formation of ternary complexes could decrease the sum of the enzyme control coefficients to below one. Experimental studies have recently confirmed this identification, as well as theoretically predicted values for the total control. Macromolecular crowding was shown to be a major candidate for the factor that modulates the non-ideal behaviour of the PTS pathway and the sum of the enzyme control coefficients.     

Probing the structure of complex macromolecular interactions by homolog specificity scanning: the P1 and P7 plasmid partition systems.

The P1 plasmid partition locus, P1 par, actively distributes plasmid copies to Escherichia coli daughter cells. It encodes two DNA sites and two proteins, ParA and ParB. Plasmid P7 uses a similar system, but the key macromolecular interactions are species specific. Homolog specificity scanning (HSS) exploits such specificities to map critical contact points between component macromolecules. The ParA protein contacts the par operon operator for operon autoregulation, and the ParB contacts the parS partition site during partition. Here, we refine the mapping of these contacts and extend the use of HSS to map protein-protein contacts. We found that ParB participates in autoregulation at the operator site by making a specific contact with ParA. Similarly, ParA acts in partition by making a specific contact with ParB bound at parS. Both these interactions involve contacts between a C-terminal region of ParA and the extreme N-terminus of ParB. As a single type of ParA-ParB complex appears to be involved in recognizing both DNA sites, the operator and the parS sites may both be occupied by a single protein complex during partition. The general HSS strategy may aid in solving the three-dimensional structures of large complexes of macromolecules.     

Effects of acid stress on polyamine levels,ion efflux,protective enzymes and macromolecular synthesis in cereal leaves

Wheat or oat leaf segments were floated on acid solutions and the changes in several parameters measured. The putrescine content of wheat leaves was 12.8-fold greater at pH 3.5 than at pH 6 after 72 h. The increase in putrescine was accompanied by an increase in the amount of K⁺ efflux from the leaf tissue to the external solution. The activities of superoxide dismutase and catalase of wheat leaves at pH 3.5 decreased to 51% and 40%, respectively, of their levels at pH 6 by 24 h. [³H]Uridine and [³H]leucine incorporation into macromolecules in oat leaves (with the lower epidermis removed) decreased to 58% and 28% of the control in response to acid stress in 8 h, at the same time as a 5-fold increase in putrescine. When DL--difluoro-methyarginine was added to the pH 3.5 buffered solution, the effect of acid was slightly less, with the incorporation into macromolecules being 64% and 35% of the pH 6 control. The results indicated that the putrescine accumulation under acid stress was concomitant with ion efflux, and a decrease in both macromolecular synthesis and the activities of superoxide dismutase and catalase in cereal leaves.Abbreviations Put putrescine - Spd spermidine - Spm spermine - SOD superoxide dismutase - CAT catalase - ADC arginine decarboxylase - DFMA DL--difluoromethylarginine This paper is dedicated to Dr. A.W. Galston in honor for his great achievements and in recognition of his continuing contributions to the study of plant science.     

Comparative electrostatic analysis of adenylyl cyclase for isoform dependent regulation properties

The enzyme adenylyl cyclase (AC) plays a pivotal role in a variety of signal transduction pathways inside the cell, where it catalyzes the cyclization of adenosine triphosphate (ATP) into the second‐messenger cyclic adenosine monophosphate (cAMP). Among other roles, AC regulates processes involved in neural plasticity, innervation of smooth muscles of the heart and the endocrine system of the pancreas. The functional diversity of AC is manifested in its different isoforms, each having a specific regulation pattern. There is an increasing amount of data available concerning the regulatory properties of AC isoforms, however little is known about the interactions on a structural level. Here, we conducted a comparative electrostatic analysis of the catalytic domains of all nine transmembrane AC isoforms with the aim of detecting, verifying and predicting the binding sites of molecular regulators on AC. The results provide support for the positioning of the binding site of the inhibitory protein G_iα at a pseudo‐symmetric position to the stimulatory G_sα binding site. They also provide a structural interpretation of the Gβγ interaction with ACs 2, 4, and 7 and suggest a new binding site for RGS2. Comparison of the small molecule binding sites on AC shows that overall they have high electrostatic similarity, but regions of electrostatic differences are identified. These could provide a basis for the development of novel compounds with isoform‐specific modulatory effects on AC. Proteins 2016; 84:1844–1858. © 2016 Wiley Periodicals, Inc.     

The Structure of a Type 3 Secretion System (T3SS) Ruler Protein Suggests a Molecular Mechanism for Needle Length Sensing

The type 3 secretion system (T3SS) and the bacterial flagellum are related pathogenicity-associated appendages found at the surface of many disease-causing bacteria. These appendages consist of long tubular structures that protrude away from the bacterial surface to interact with the host cell and/or promote motility. A proposed “ruler” protein tightly regulates the length of both the T3SS and the flagellum, but the molecular basis for this length control has remained poorly characterized and controversial. Using the Pseudomonas aeruginosa T3SS as a model system, we report the first structure of a T3SS ruler protein, revealing a “ball-and-chain” architecture, with a globular C-terminal domain (the ball) preceded by a long intrinsically disordered N-terminal polypeptide chain. The dimensions and stability of the globular domain do not support its potential passage through the inner lumen of the T3SS needle. We further demonstrate that a conserved motif at the N terminus of the ruler protein interacts with the T3SS autoprotease in the cytosolic side. Collectively, these data suggest a potential mechanism for needle length sensing by ruler proteins, whereby upon T3SS needle assembly, the ruler protein''s N-terminal end is anchored on the cytosolic side, with the globular domain located on the extracellular end of the growing needle. Sequence analysis of T3SS and flagellar ruler proteins shows that this mechanism is probably conserved across systems.     

Xist nucleates local protein gradients to propagate silencing across the X chromosome

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