Coordinating Organismal Metabolism During Protein Misfolding in the ER Through the Unfolded Protein Response.

The endoplasmic reticulum (ER) is a cellular organelle responsible for folding of secretory and membrane proteins. Perturbance in ER homeostasis caused by various intrinsic/extrinsic stimuli challenges the protein-folding capacity of the ER, leading to an ER dysfunction, called ER stress. Cells have developed a defensive response to adapt and/or survive in the face of ER stress that may be detrimental to cell function and survival. When exposed to ER stress, the cell activates a complex and elaborate signaling network that includes translational modulation and transcriptional induction of genes. In addition to these autonomous responses, recent studies suggest that the stressed tissue secretes peptides or unknown factors that transfer the signal to other cells in the same or different organs, leading the organism as a whole to cope with challenges in a non-autonomous manner. In this review, we discuss the mechanisms by which cells adapt to ER stress challenges autonomously and transfer the stress signal to non-stressed cells in different organs.

Conformationally organized lysine isosteres in Streptococcus pyogenes M protein mediate direct high-affinity binding to human plasminogen.

The binding of human plasminogen (hPg) to the surface of the human pathogen group A Streptococcus pyogenes (GAS) and subsequent hPg activation to the protease plasmin generate a proteolytic surface that GAS employs to circumvent host innate immunity. Direct high-affinity binding of hPg/plasmin to pattern D GAS is fully recapitulated by the hPg kringle 2 domain (K2hPg) and a short internal peptide region (a1a2) of a specific subtype of bacterial surface M protein, present in all GAS pattern D strains. To better understand the nature of this binding, critical to the virulence of many GAS skin-tropic strains, we used high-resolution NMR to define the interaction of recombinant K2hPg with recombinant a1a2 (VKK38) of the M protein from GAS isolate NS455. We found a 2:1 (m/m) binding stoichiometry of K2hPg/VKK38, with the lysine-binding sites of two K2hPg domains anchored to two regions of monomeric VKK38. The K2hPg/VKK38 binding altered the VKK38 secondary structure from a helical apo-peptide with a flexible center to an end-to-end K2hPg-bound α-helix. The K2hPg residues occupied opposite faces of this helix, an arrangement that minimized steric clashing of K2hPg We conclude that VKK38 provides two conformational lysine isosteres that each interact with the lysine-binding sites in K2hPg Further, the adoption of an α-helix by VKK38 upon binding to K2hPg sterically optimizes the side chains of VKK38 for maximal binding to K2hPg and minimizes steric overlap between the K2hPg domains. The mechanism for hPg/M protein binding uncovered here may facilitate targeting of GAS virulence factors for disease management.

Direct Host Plasminogen Binding to Bacterial Surface M-protein in Pattern D Strains of Streptococcus pyogenes Is Required for Activation by Its Natural Coinherited SK2b Protein.

Streptokinase (SK), secreted by Group A Streptococcus (GAS), is a single-chain ∼47-kDa protein containing three consecutive primary sequence regions that comprise its α, ß, and γ modules. Phylogenetic analyses of the variable ß-domain sequences from different GAS strains suggest that SKs can be arranged into two clusters, SK1 and SK2, with a subdivision of SK2 into SK2a and SK2b. SK2b is secreted by skin-tropic Pattern D M-protein strains that also express plasminogen (human Pg (hPg)) binding Group A streptococcal M-protein (PAM) as its major cell surface M-protein. SK2a-expressing strains are associated with nasopharynx tropicity, and many of these strains express human fibrinogen (hFg) binding Pattern A-C M-proteins, e.g. M1. PAM interacts with hPg directly, whereas M1 binds to hPg indirectly via M1-bound hFg. Subsequently, SK is secreted by GAS and activates hPg to plasmin (hPm), thus generating a proteolytic surface on GAS that enhances its dissemination. Due to these different modes of hPg/hPm recognition by GAS, full characterizations of the mechanisms of activation of hPg by SK2a and SK2b and their roles in GAS virulence are important topics. To more fully examine these subjects, isogenic chimeric SK- and M-protein-containing GAS strains were generated, and the virulence of these chimeric strains were analyzed in mice. We show that SK and M-protein alterations influenced the virulence of GAS and were associated with the different natures of hPg activation and hPm binding. These studies demonstrate that GAS virulence can be explained by disparate hPg activation by SK2a and SK2b coupled with the coinherited M-proteins of these strains.

A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase.

The nuclear phosphoinositide response to stress.

Accumulating evidence reveals that nuclear phosphoinositides (PIs) serve as central signaling hubs that control a multitude of nuclear processes by regulating the activity of nuclear proteins. In response to cellular stressors, PIs accumulate in the nucleus and multiple PI isomers are synthesized by the actions of PI-metabolizing enzymes, kinases, phosphatases and phospholipases. By directly interacting with effector proteins, phosphoinositide signals transduce changes in cellular functions. Here we describe nuclear phosphoinositide signaling in multiple sub-nuclear compartments and summarize the literature that demonstrates roles for specific kinases, phosphatases, and phospholipases in the orchestration of nuclear phosphoinositide signaling in response to cellular stress. Additionally, we discuss the specific PI-protein complexes through which these lipids execute their functions by regulating the configuration, stability, and transcription activity of their effector proteins. Overall, our review provides a detailed landscape of the current understanding of the nuclear PI-protein interactome and its role in shaping the coordinated response to cellular stress.