25 research outputs found
Surviving Endoplasmic Reticulum Stress Is Coupled to Altered Chondrocyte Differentiation and Function
In protein folding and secretion disorders, activation of endoplasmic reticulum (ER) stress signaling (ERSS) protects cells, alleviating stress that would otherwise trigger apoptosis. Whether the stress-surviving cells resume normal function is not known. We studied the in vivo impact of ER stress in terminally differentiating hypertrophic chondrocytes (HCs) during endochondral bone formation. In transgenic mice expressing mutant collagen X as a consequence of a 13-base pair deletion in Col10a1 (13del), misfolded α1(X) chains accumulate in HCs and elicit ERSS. Histological and gene expression analyses showed that these chondrocytes survived ER stress, but terminal differentiation is interrupted, and endochondral bone formation is delayed, producing a chondrodysplasia phenotype. This altered differentiation involves cell-cycle re-entry, the re-expression of genes characteristic of a prehypertrophic-like state, and is cell-autonomous. Concomitantly, expression of Col10a1 and 13del mRNAs are reduced, and ER stress is alleviated. ERSS, abnormal chondrocyte differentiation, and altered growth plate architecture also occur in mice expressing mutant collagen II and aggrecan. Alteration of the differentiation program in chondrocytes expressing unfolded or misfolded proteins may be part of an adaptive response that facilitates survival and recovery from the ensuing ER stress. However, the altered differentiation disrupts the highly coordinated events of endochondral ossification culminating in chondrodysplasia
Autologous transplantation of genetically modified iris pigment epithelial cells: A promising concept for the treatment of age-related macular degeneration and other disorders of the eye
Age-related macular degeneration (ARMD) is the leading cause for visual impairment and blindness in the elder population. Laser photocoagulation, photodynamic therapy and excision of neovascular membranes have met with limited success. Submacular transplantation of autologous iris pigment epithelial (IPE) cells has been proposed to replace the damaged retinal pigment epithelium following surgical removal of the membranes. We tested our hypothesis that the subretinal transplantation of genetically modified autologous IPE cells expressing biological therapeutics might be a promising strategy for the treatment of ARMD and other retinal disorders. Pigment epithelium-derived factor (PEDF) has strong antiangiogenic and neuroprotective activities in the eye. Subretinal transplantation of PEDF expressing IPE cells inhibited pathological choroidal neovascularization in rat models of laser-induced rupture of Bruch's membrane and of oxygen induced ischemic retinopathy. PEDF expressing IPE transplants also increased the survival and preserved rhodopsin expression of photoreceptor cells in the RCS rat, a model of retinal degeneration. These findings suggest a promising concept for the treatment of ARMD and other retinal disorders
Mutations Targeting Intermodular Interfaces or Calcium Binding Destabilize the Thrombospondin-2 Signature Domain*
Thrombospondins (THBSs) are a family of secreted calcium-binding
glycoproteins with roles in angiogenesis, cell motility, apoptosis,
cytoskeletal organization, and extracellular matrix organization. The THBS-2
signature domain (three epidermal growth factor (EGF)-like modules, a wire
module with 13 calcium-binding repeats, and a lectin-like module) binds 30
calcium ions and forms extensive interactions among its parts. We explored the
significance of these structural elements by examining the impact of 10
different mutations known to result in pseudoachondrodysplasia or multiple
epiphyseal dysplasia when found in the homologous wire and lectin-like modules
of thrombospondin-5 (THBS-5). A variety of observations indicate that the
mutations result in unstable THBS-5 proteins that aggregate in the endoplasmic
reticulum. We introduced the mutations into homologous sites of a THBS-2
construct, for which the crystal structure is known, and determined the
effects of the mutations on structure as assayed by differential scanning
calorimetry and expression of the epitope for the 4B6.13
conformation-sensitive antibody. Abnormalities were found in one or more of
several readouts: stability of interactions between the wire and lectin-like
modules, stabilities of the EGF-like and wire modules, expression of the
4B6.13 epitope in soluble protein, and expression of the 4B6.13 epitope in
substrate-adsorbed protein at different calcium concentrations. The patterns
of abnormalities support the idea that the EGF-like, wire, and lectin-like
modules constitute a dynamic and interactive calcium-sensitive structure in
which a distortion at one site is transmitted to distal sites, leading to
global changes in the protein
Stereospecific Suzuki, Sonogashira, and Negishi Coupling Reactions of <i>N</i>‑Alkoxyimidoyl Iodides and Bromides
A high-yielding
stereospecific route to the synthesis of single
geometric isomers of diaryl oxime ethers through Suzuki coupling of <i>N</i>-alkoxyimidoyl iodides is described. This reaction occurs
with complete retention of the imidoyl halide geometry to give single <i>E</i>- or <i>Z</i>-isomers of diaryl oxime ethers.
The Sonogashira coupling of <i>N</i>-alkoxyimidoyl iodides
and bromides with a wide variety of terminal alkynes to afford single
geometric isomers of aryl alkynyl oxime ethers has also been developed.
Several of these reactions proceed through copper-free conditions.
The Negishi coupling of <i>N</i>-alkoxyimidoyl halides is
introduced. The <i>E</i> and <i>Z</i> configurations
of nine Suzuki-coupling products and two Sonogashira-coupling products
were confirmed by X-ray crystallography
Stereospecific Suzuki, Sonogashira, and Negishi Coupling Reactions of <i>N</i>‑Alkoxyimidoyl Iodides and Bromides
A high-yielding
stereospecific route to the synthesis of single
geometric isomers of diaryl oxime ethers through Suzuki coupling of <i>N</i>-alkoxyimidoyl iodides is described. This reaction occurs
with complete retention of the imidoyl halide geometry to give single <i>E</i>- or <i>Z</i>-isomers of diaryl oxime ethers.
The Sonogashira coupling of <i>N</i>-alkoxyimidoyl iodides
and bromides with a wide variety of terminal alkynes to afford single
geometric isomers of aryl alkynyl oxime ethers has also been developed.
Several of these reactions proceed through copper-free conditions.
The Negishi coupling of <i>N</i>-alkoxyimidoyl halides is
introduced. The <i>E</i> and <i>Z</i> configurations
of nine Suzuki-coupling products and two Sonogashira-coupling products
were confirmed by X-ray crystallography
Structure of the Calcium-Rich Signature Domain of Human Thrombospondin-2
Thrombospondins (TSPs) are secreted glycoproteins that play key roles in interactions between cells and the extracellular matrix. Here, we describe the 2.6 Ă… resolution crystal structure of the glycosylated signature domain of human TSP-2, which includes three epidermal growth factor-like (EGF-like) modules, 13 aspartate-rich repeats, and a lectin-like module. These elements interact extensively to form three striking structural regions termed the stalk, wire, and globe. The TSP-2 signature domain is stabilized by these interactions and by a network of 30 bound Ca2+ ions and 18 disulfide bonds. The structure suggests how genetic alterations of TSPs result in disease