The gain-of-function mutation in the TALK-1 K
+
channel (p.L114P) is associated with maturity-onset diabetes of the young (MODY). TALK-1 is a key regulator of β-cell electrical activity and ...glucose-stimulated insulin secretion. The
KCNK16
gene encoding TALK-1 is the most abundant and β-cell-restricted K
+
channel transcript. To investigate the impact of
KCNK16
L114P on glucose homeostasis and confirm its association with MODY, a mouse model containing the
Kcnk16
L114P mutation was generated. Heterozygous and homozygous
Kcnk16
L114P mice exhibit increased neonatal lethality in the C57BL/6J and the CD-1 (ICR) genetic background, respectively. Lethality is likely a result of severe hyperglycemia observed in the homozygous
Kcnk16
L114P neonates due to lack of glucose-stimulated insulin secretion and can be reduced with insulin treatment.
Kcnk16
L114P increased whole-cell β-cell K
+
currents resulting in blunted glucose-stimulated Ca
2+
entry and loss of glucose-induced Ca
2+
oscillations. Thus, adult
Kcnk16
L114P mice have reduced glucose-stimulated insulin secretion and plasma insulin levels, which significantly impairs glucose homeostasis. Taken together, this study shows that the MODY-associated
Kcnk16
L114P mutation disrupts glucose homeostasis in adult mice resembling a MODY phenotype and causes neonatal lethality by inhibiting islet insulin secretion during development. These data suggest that TALK-1 is an islet-restricted target for the treatment for diabetes.
Abstract
Background
Staphylococcus aureus is the most common invasive bacterial pathogen in children. The constant evolution of S. aureus is a barrier to the development of successful interventions. ...A detailed understanding of the current molecular epidemiology of actively circulating, invasive S. aureus strains and a comparison to both colonization strains and historical invasive strains to determine which virulence factors and genetic lineages are preserved over time are important unmet needs in this field.
Methods
We compared four groups of S. aureus isolates: 45 invasive strains obtained during the epidemic methicillin-resistance S. aureus (MRSA) peak (2010-2012); 74 invasive contemporary strains (2015-2021); 61 colonizing strains from 2004; and 30 colonizing strains from 2021. Genetic relatedness (via clonal complex typing) and virulence factor presence were determined via whole genome sequencing.
Results
Among invasive isolates, the prevalence of clonal complex 8 (CC8) decreased significantly over time from 87% to 46% among typeable strains (p< 0.05, Fig.1). Similarly, the presence of Panton-Valentine leucocidin (PVL) decreased from 71% to 40% (p< 0.01). 56% of invasive isolates from 2010-2012 were MRSA, vs 37% from 2015-2021 (p= 0.05, Fig. 2). In contrast to PVL, the presence of leukocidins LukED and LukAB remained stably prevalent over time at 84% and 100% (Fig. 3). Colonization isolates overall remained diverse, with no dominant clonal complex (Fig. 1). Figure 1Figure 2Figure 3
Conclusion
Invasive S. aureus molecular epidemiology has changed significantly over the past decade. The former dominance of CC8 and PVL-positive MRSA strains has shifted to a more diverse, predominantly MSSA group of contemporary strains. Other virulence factors (LukED, LukAB) have remained prevalent among invasive strains regardless of genetic lineage. Targets of intervention should be those that are preserved throughout the evolution of this major human pathogen.
Disclosures
All Authors: No reported disclosures.
The functional hallmark of annexins is the ability to bind to the surface of phospholipid membranes in a reversible, Ca2+-dependent manner. We now report that human annexin V and hydra annexin XII ...reversibly bound to phospholipid vesicles in the absence of Ca2+ at low pH; half-maximal vesicle association occurred at pH 5.3 and 5.8, respectively. The following biochemical data support the hypothesis that these annexins insert into bilayers at mildly acidic pH. First, a photoactivatable reagent (3-trifluoromethyl)-3-(m-125Iiodophenyl)diazirine) which selectively labels proteins exposed to the hydrophobic domain of bilayers reacted with these annexins at pH 5.0 and below but not at neutral pH. Second, in a Triton X-114 partitioning assay, annexins V and XII act as integral membrane proteins at low pH and as hydrophilic proteins at neutral pH; in the presence of phospholipids half-maximal partitioning into detergent occurred at pH ≈ 5.0. Finally, annexin V or XII formed single channels in phospholipid bilayers at low pH but not at neutral pH. A model is discussed in which the concentrations of H+ and Ca2+ regulate the reversible conversion of three forms of annexinssoluble, peripheral membrane, and transmembrane.
The detailed structural basis for the cryptic nature (crypticity) of a B cell epitope harbored by an autoantigen is unknown. Because the immune system may be ignorant of the existence of such ...“cryptic” epitopes, their exposure could be an important feature in autoimmunity. Here we investigated the structural basis for the crypticity of the epitopes of the Goodpasture autoantigen, the α3α4α5 noncollagenous-1 (NC1) hexamer, a globular domain that connects two triple-helical molecules of the α3α4α5 collagen IV network. The NC1 hexamer occurs in two isoforms as follows: the M-isoform composed of monomer subunits in which the epitopes are accessible to autoantibodies, and the D-isoform composed of both monomer and dimer subunits in which the epitopes are cryptic. The D-isoform was characterized with respect to quaternary structure, as revealed by mass spectrometry of dimer subunits, homology modeling, and molecular dynamics simulation. The results revealed that the D-isoform contains two kinds of cross-links as follows: S-hydroxylysyl-methionine and S-lysyl-methionine cross-links, which stabilize the α3α5-heterodimers and α4α4-homodimers, respectively. Construction and analysis of a three-dimensional model of the D-isoform of the α3α4α5 NC1 hexamer revealed that crypticity is a consequence of the following: (a) sequestration of key residues between neighboring subunits that are stabilized by domain-swapping interactions, and (b) by cross-linking of subunits at the trimer-trimer interface, which stabilizes the structural integrity of the NC1 hexamer and protects against binding of autoantibodies. The sequestrated epitopes and cross-linked subunits represent a novel structural mechanism for conferring immune privilege at the level of quaternary structure. Perturbation of the quaternary structure may be a key factor in the etiology of Goodpasture disease.
Mechanism of Chain Selection in the Assembly of Collagen IV Khoshnoodi, Jamshid; Sigmundsson, Kristmundur; Cartailler, Jean-Philippe ...
Journal of biological chemistry/The Journal of biological chemistry,
03/2006, Volume:
281, Issue:
9
Journal Article
Peer reviewed
Open access
Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and ...assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the α1(IV) and α2(IV) NC1 domains. We show that the differential affinity of α2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the α1(IV) NC1 domain, we conclude that the α2(IV) chain plays a regulatory role in directing chain composition in the assembly of (α1)2α2 triple-helical molecule. Detailed crystal structure analysis of the (α1)2α22 NC1 hexamer and sequence alignments of the NC1 domains of all six α-chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a β-hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.
Collagens comprise a large superfamily of extracellular matrix proteins that play diverse roles in tissue function. The mechanism by which newly synthesized collagen chains recognize each other and ...assemble into specific triple-helical molecules is a fundamental question that remains unanswered. Emerging evidence suggests a role for the non-collagenous domain (NC1) located at the C-terminal end of each chain. In this study, we have investigated the molecular mechanism underlying chain selection in the assembly of collagen IV. Using surface plasmon resonance, we have determined the kinetics of interaction and assembly of the alpha 1(IV) and alpha 2(IV) NC1 domains. We show that the differential affinity of alpha 2(IV) NC1 domain for dimer formation underlies the driving force in the mechanism of chain discrimination. Given its characteristic domain recognition and affinity for the alpha 1(IV) NC1 domain, we conclude that the alpha 2(IV) chain plays a regulatory role in directing chain composition in the assembly of ( alpha 1) sub(2) alpha 2 triple-helical molecule. Detailed crystal structure analysis of the ( alpha 1) sub(2) alpha 2 sub(2) NC1 hexamer and sequence alignments of the NC1 domains of all six alpha -chains from mammalian species revealed the residues involved in the molecular recognition of NC1 domains. We further identified a hypervariable region of 15 residues and a beta -hairpin structural motif of 13 residues as two prominent regions that mediate chain selection in the assembly of collagen IV. To our knowledge, this report is the first to combine kinetics and structural data to describe molecular basis for chain selection in the assembly of a collagen molecule.
We used mice lacking
, a key component of the β-cell K
-channel, to analyze the effects of a sustained elevation in the intracellular Ca
concentration (Ca
) on β-cell identity and gene expression. ...Lineage tracing analysis revealed the conversion of β-cells lacking
into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified
β-cells confirmed an increase in
gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca
signaling, the maintenance of β-cell identity, and cell adhesion. The expression of
and
, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in Ca
Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which,
, was confirmed to be directly controlled by Ca
influx in β-cells. Interestingly, among the upregulated genes is
, a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell Ca
in
islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca
-regulated genes.
Global Structural Changes in Annexin 12 Isas, J.Mario; Patel, Darshana R.; Jao, Christine ...
Journal of biological chemistry/The Journal of biological chemistry,
08/2003, Volume:
278, Issue:
32
Journal Article
Peer reviewed
Open access
Ca2+-dependent membrane interaction has long been recognized as a general property of the annexin (ANX) family of proteins. More recently, it has become clear that ANXs can also undergo ...Ca2+-independent membrane interactions at mildly acidic pH. Here we use site-directed spin labeling in combination with circular dichroism and biochemical labeling methods to compare the structure and membrane topography of these two different membrane-bound forms of ANX12. Our results reveal strong similarities between the solution structure and the structure of the Ca2+-dependent membrane-bound form at neutral pH. In contrast, all Ca2+-independent membrane interactions tested resulted in large scale conformational changes and membrane insertion. Pairs of spin labels that were in close proximity across the interface of different domains of the protein in both the soluble and Ca2+-dependent membrane form were >25 Å apart in the Ca2+-independent membrane-bound form. Despite these major conformational changes, the overall secondary structure content did not appear to be strongly altered and ANX12 remained largely helical. Thus, Ca2+-independent membrane interaction leads to massive refolding but not unfolding. Refolding did not occur at low pH in the absence of membranes but occurred within a few seconds after phospholipid vesicles were added. The phospholipid composition of the vesicles was an important modulator of Ca2+-independent membrane interaction. For example, cardiolipin-containing vesicles induced Ca2+-independent membrane interaction even at near neutral pH, thereby raising the possibility that lipid composition could induce relatively rapid Ca2+-independent membrane interaction in vivo.
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