A highly multiplexed superresolution imaging strategy with single-molecule accuracy enabled by fluorescent nanodiamonds called madSTORM affords the ability to define spatial relationships among constituent molecules within structures. It makes it possible to probe the molecular topology of complex signaling cascades and other heterogeneous networks.

Human La protein has been implicated in facilitating internal ribosome entry site (IRES)-mediated translation of hepatitis C virus (HCV).The mechanism of internal initiation of translation of hepatitis C virus (HCV) RNA is unique and fundamentally different from the cap-dependent translation of host cell mRNAs, and thus, the HCV internal ribosome entry site (IRES) offers a potential target for developing novel antiviral therapeutics (6).The ribosome assembly at the HCV IRES has been shown to be prokaryotic-like and requires a minimal number of initiation factors (23). The HCV IRES has been shown to be capable of binding directly to 40S ribosomal subunit with the help of the ribosomal protein S5 (10, 18). Although, HCV IRES binds to the 40S ribosomal subunit specifically and stably even in the absence of any initiation factors, efficient translation requires some of the canonical initiation factors and noncanonical trans-acting factors, possibly to facilitate ribosome binding and to ensure proper positioning of the initiator AUG (iAUG) codon in the P site (13). Several cellular trans-acting factors have been reported to be critically required for HCV IRES-mediated translation, which includes human La autoantigen (2).Human La protein was originally identified in the sera from patients with systemic lupus erythematosus and Sjögren's syndrome (28). Earlier, it was reported in the literature that La protein contains three RNA recognition motifs (RRMs), which were putatively located between residues 1 and 100 (previously named RRM1), 101 and 208 (previously, RRM2), and 209 and 300 (previously, RRM3) (12, 24). However, according to the recent nomenclature, the first structured domain in human La is termed "La motif" located between residues 16 and 75 ], followed by two RRMs, RRM (112-184) and RRM (230-300) (22,29). Although, based on the structure determinations, the precise boundaries of the structured cores were found to be slightly different , RRM (229-327)], they largely encompass the above regions (1, 17). La becomes associated with the 3Ј termini of many newly synthesized small RNAs made by RNA polymerase III, as well as certain small RNAs synthesized by other RNA polymerases (20,31). The N terminal 80 amino acid (aa) residues termed the La motif and the adjacent RRM part have been shown to be required for high-affinity binding with polymerase III transcripts, where the La motif helps in specific recognition for the polyuridylate (UUU OH ) sequence at the 3Ј end of the RNA. The C-terminal RRM (230-300) has been shown to have a beta sheet comprising five strands and a long C-terminal helix that binds to the putative RNA binding site (17). The central RRM (112-184) has been shown to possess a classical RRMtype fold containing four stranded beta sheets backed by two alpha helices. It also has an additional beta strand inserted between alpha 2 and beta 4. The helix alpha 3 sheet of RRM (112-184) is predominantly hydrophilic and protrudes away

The T cell antigen receptor encounters foreign antigen during the immune response. Receptor engagement leads to activation of specific protein tyrosine kinases, which then phosphorylate multiple enzymes and adapter proteins. One such enzyme, phospholipase-Cγ1, is responsible for cleavage of a plasma membrane lipid substrate, a phosphoinositide, into two second messengers, diacylglycerol, which activates several enzymes including protein kinase C, and an inositol phosphate, which induces intracellular calcium elevation. In T cells, phospholipase-Cγ1 is recruited to the plasma membrane as part of a four-protein complex containing three adapter molecules. We have used recombinant proteins and synthetic phosphopeptides to reconstitute this quaternary complex in vitro. Extending biophysical tools to study concurrent interactions of the four protein components, we demonstrated the formation and determined the composition of the quaternary complex using multisignal analytical ultracentrifugation, and we characterized the thermodynamic driving forces of assembly by isothermal calorimetry. We demonstrate that the four proteins reversibly associate in a circular arrangement of binding interfaces, each protein interacting with two others. Three interactions are of high affinity, and the fourth is of low affinity, with the assembly of the quaternary complex exhibiting significant enthalpy-entropy compensation as in an entropic switch. Formation of this protein complex enables subsequent recruitment of additional molecules needed to activate phospholipase-Cγ1. Understanding the formation of this complex is fundamental to full characterization of a central pathway in T cell activation. Such knowledge is critical to developing ways in which this pathway can be selectively inhibited.phospholipase C | protein complex | signal transduction | T cell antigen receptor | tyrosine phosphorylation T hree decades of investigation have resulted in a description of T cell antigen receptor (TCR) engagement and the biochemical events that follow. The binding of the TCR ligand, an antigenic peptide presented by a protein encoded by the major histocompatibility complex, leads to the activation of associated and recruited protein tyrosine kinases (PTKs) (1). Critical substrates of these PTKs include the adapter molecules, linker for activation of T cells (LAT), and Src homology 2 (SH2) domaincontaining leukocyte protein of 76kDa (SLP-76) (2, 3). The phosphorylation of tyrosine residues on these adapters initiates binding of many other adapters and enzymes to generate molecular complexes of great heterogeneity (4). In this manner enzymes such as phospholipase C-γ1 (PLC-γ1) and son of sevenless homolog 1 (SOS1) are recruited to the plasma membrane where they encounter their substrates. The products that are a consequence of these enzyme activations-diacylglycerol, elevated intracellular calcium, and activated Ras-have complex intracellular effects during the process of T cell activation (5, 6).Sites of protein-protein interactions between these v...