Increased dynamics in the 40-57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation

Thrombocytopenia 4 is an inherited autosomal dominant thrombocytopenia, which occurs due to mutations in the human gene for cytochrome c that results in enhanced mitochondrial apoptotic activity. The Gly41Ser mutation was the first to be reported. Here we report stopped-flow kinetic studies of azide binding to human ferricytochrome c and its Gly41Ser variant, together with backbone amide H/D exchange and 15N-relaxation dynamics using NMR spectroscopy, to show that alternative conformations are kinetically and thermodynamically more readily accessible for the Gly41Ser variant than for the wild-type protein. Our work reveals a direct conformational link between the 40-57 Ω-loop in which residue 41 resides and the dynamical properties of the axial ligand to the heme iron, Met80, such that the replacement of glycine by serine promotes the dissociation of the Met80 ligand, thereby increasing the population of a peroxidase active state, which is a key non-native conformational state in apoptosis

International union of basic and clinical pharmacology. CVIII. Calcium-sensing receptor nomenclature, pharmacology, and function

The calcium-sensing receptor (CaSR) is a class C G protein-coupled receptor that responds to multiple endogenous agonists and allosteric modulators, including divalent and trivalent cations, L-amino acids, <i>γ</i>-glutamyl peptides, polyamines, polycationic peptides, and protons. The CaSR plays a critical role in extracellular calcium (Ca<sup>2+</sup> <sub>o</sub>) homeostasis, as demonstrated by the many naturally occurring mutations in the CaSR or its signaling partners that cause Ca<sup>2+</sup> <sub>o</sub> homeostasis disorders. However, CaSR tissue expression in mammals is broad and includes tissues unrelated to Ca<sup>2+</sup> <sub>o</sub> homeostasis, in which it, for example, regulates the secretion of digestive hormones, airway constriction, cardiovascular effects, cellular differentiation, and proliferation. Thus, although the CaSR is targeted clinically by the positive allosteric modulators (PAMs) cinacalcet, evocalcet, and etelcalcetide in hyperparathyroidism, it is also a putative therapeutic target in diabetes, asthma, cardiovascular disease, and cancer. The CaSR is somewhat unique in possessing multiple ligand binding sites, including at least five putative sites for the "orthosteric" agonist Ca<sup>2+</sup> <sub>o</sub>, an allosteric site for endogenous L-amino acids, two further allosteric sites for small molecules and the peptide PAM, etelcalcetide, and additional sites for other cations and anions. The CaSR is promiscuous in its G protein-coupling preferences, and signals via G<sub>q/11</sub>, G<sub>i/o</sub>, potentially G<sub>12/13</sub>, and even G<sub>s</sub> in some cell types. Not surprisingly, the CaSR is subject to biased agonism, in which distinct ligands preferentially stimulate a subset of the CaSR's possible signaling responses, to the exclusion of others. The CaSR thus serves as a model receptor to study natural bias and allostery. SIGNIFICANCE STATEMENT: The calcium-sensing receptor (CaSR) is a complex G protein-coupled receptor that possesses multiple orthosteric and allosteric binding sites, is subject to biased signaling via several different G proteins, and has numerous (patho)physiological roles. Understanding the complexities of CaSR structure, function, and biology will aid future drug discovery efforts seeking to target this receptor for a diversity of diseases. This review summarizes what is known to date regarding key structural, pharmacological, and physiological features of the CaSR

Molecular basis for universal HLA-A*0201–restricted CD8+ T-cell immunity against influenza viruses

Memory CD8+ T lymphocytes (CTLs) specific for antigenic peptides derived from internal viral proteins confer broad protection against distinct strains of influenza A virus (IAV). However, immune efficacy can be undermined by the emergence of escape mutants. To determine how T-cell receptor (TCR) composition relates to IAV epitope variability, we used ex vivo peptide-HLA tetramer enrichment and single-cell multiplex analysis to compare TCRs targeted to the largely conserved HLA-A∗0201-M158 and the hypervariable HLA-B∗3501-NP418 antigens. The TCRαβs for HLA-B∗3501-NP418+ CTLs varied among individuals and across IAV strains, indicating that a range of mutated peptides will prime different NP418-specific CTL sets. Conversely, a dominant public TRAV27/TRBV19+ TCRαβ was selected in HLA-A∗0201+ donors responding to M158. This public TCR cross-recognized naturally occurring M158 variants complexed with HLA-A∗0201. Ternary structures showed that induced-fit molecular mimicry underpins TRAV27/TRBV19+ TCR specificity for the WT and mutant M158 peptides, suggesting the possibility of universal CTL immunity in HLA-A∗0201+ individuals. Combined with the high population frequency of HLA-A∗0201, these data potentially explain the relative conservation of M158. Moreover, our results suggest that vaccination strategies aimed at generating broad protection should incorporate variant peptides to elicit cross-reactive responses against other specificities, especially those that may be relatively infrequent among IAV-primed memory CTLs

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4–14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition

Backbone resonance assignments of ferric human cytochrome c and the pro-apoptotic G41S mutant in the ferric and ferrous states

Human cytochrome c is a multi-functional protein with key roles in both the mitochondrial electron transfer chain and in apoptosis. In the latter, a complex formed between the mitochondrial phospholipid cardiolipin and cytochrome c is crucial for instigating the release of pro-apoptotic factors, including cytochrome c, from the mitochondrion into the cytosol. The G41S mutant of human cytochrome c is the only known disease-related variant of cytochrome c and causes increased apoptotic activity in patients with autosomal dominant thrombocytopenia. NMR spectroscopy can be used to investigate the interaction of human cytochrome c with cardiolipin and the structural and dynamic factors, which may contribute to enhanced apoptotic activity for the G41S mutant. We present here essentially full backbone amide resonance assignments for ferric human cytochrome c (98 %) as well as assignments of both the ferric (92 %) and ferrous (95 %) forms of the G41S mutant. Backbone amide chemical shift differences between the wild type and G41S mutant in the ferric state reveals significant changes around the mutation site, with many other amides also affected. This suggests the possibility of increased dynamics and/or a change in the paramagnetic susceptibility tensor of the G41S mutant relative to the wild type protein

Progressive Supranuclear Palsy and Corticobasal Degeneration.

Progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) are neurodegenerative tauopathies with neuronal and glial lesions composed of tau that is composed predominantly of isomers with four repeats in the microtubule-binding domain (4R tau). The brain regions vulnerable to pathology in PSP and CBD overlap, but there are differences, particularly with respect to distribution of neuronal loss, the relative abundance of neuronal and glial lesions, the morphologic features of glial lesions, and the frequency of comorbid pathology. Both PSP and CBD have a wide spectrum of clinical manifestations, including disorders of movement and cognition. Recognition of phenotypic diversity in PSP and CBD may improve antemortem diagnostic accuracy, which tends to be very good for the most common presentation of PSP (Richardson syndrome), but poor for the most characteristic presentation of CBD (corticobasal syndrome: CBS). Development of molecular and imaging biomarkers may improve antemortem diagnostic accuracy. Currently, multidisciplinary symptomatic and supportive treatment with pharmacological and non-pharmacological strategies remains the standard of care. In the future, experimental therapeutic trials will be important to slow disease progression