HMGB1 orchestrates leukocyte recruitment and their induction to secrete inflammatory cytokines by switching between mutually exclusive redox states.

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Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn 263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an ␣ v ␤ 3 -integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGRto-isoDGR sequence transition in fibronectin fragments generates ␣ v ␤ 3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.Fibronectins are adhesive proteins that mediate a variety of cellular interactions with extracellular matrix and play important roles in hemostasis, thrombosis, inflammation, wound repair, angiogenesis, and embryogenesis (1, 2). About 20 isoforms of human fibronectin can be generated as a result of alternative splicing of the primary transcript (1, 3). Fibronectins are large glycoproteins (ϳ450 kDa) composed of two nearly identical disulfide-bonded subunits present in most body fluids and extracellular matrix of many tissues. Each subunit consists of three types of repeating homologous modules termed FN-I, FN-II, and FN-III repeats. Alternatively spliced modules, called EDA, EDB, and IIICS, can also be present (1, 3). Single modules or groups of modules may contain binding sites for different molecules, including sulfated glycosaminoglycans, DNA, gelatin, heparin, and fibrin (1, 3, 4). Furthermore, fibronectins contain binding sites for about half of the known cell surface integrin receptors (5, 6). In particular, the FN-III 10 repeat contains an RGD site that can bind, and ␣II b ␤ 3 integrins, while the FN-III 9 repeat contains the so-called "synergy site" PHSRN that cooperates with RGD in the binding of ␣ 5 ␤ 1 and ␣II b ␤ 3 (1, 7).Primary and tertiary structure analysis of human fibronectin showed that this protein contains two GNGRG loops, located in FN-I 5 and FN-I 7 modules, that are conserved in bovine, murine, rat, amphibian, and fish (8). Two additional NGR sites, less conserved, are also present in human FN-II 1 and FN-III 9 (see Fig. 1). Recent experimental work showed that peptides containing the NGR motif can inhibit ␣ 5 ␤ 1 -and ␣ v ␤ 1 -mediated cell adhesion to fibronectin (9).These notions prompted us to investigate the functional role of NGR in fibronectin. We observed that the NGR sequence of FN-I 5 (residues 263-265) promotes endothelial cell adhesion via an...

The mechanism of neurodegeneration caused by ␤-amyloid in Alzheimer disease is controversial. Neuronal toxicity is exerted mostly by various species of soluble ␤-amyloid oligomers that differ in their N-and C-terminal domains. However, abundant accumulation of ␤-amyloid also occurs in the brains of cognitively normal elderly people, in the absence of obvious neuronal dysfunction. We postulated that neuronal toxicity depends on the molecular composition, rather than the amount, of the soluble ␤-amyloid oligomers. Here we show that soluble ␤-amyloid aggregates that accumulate in Alzheimer disease are different from those of normal aging in regard to the composition as well as the aggregation and toxicity properties.A series of evidence indicates that progressive cerebral accumulation of ␤-amyloid (A␤), 2 a proteolytic product of transmembrane protein APP, is the primary pathogenic event of Alzheimer disease (AD) (1). Recent clues indicate that small, soluble A␤ aggregates produce more severe synaptic dysfunction and neuronal damage than do A␤ polymers (2-5). This behavior is common to all known pathogenic and nonpathogenic amyloidogenic peptides (6, 7). Soluble A␤ is detectable early in the cerebral cortex of subjects at risk for AD pathology, several years before the formation and deposition of amyloid fibrils (8). Hence, the analysis of soluble A␤ in brain tissue allows the characterization of the toxic form of the peptide.A strong argument against the amyloid hypothesis is the abundant and constant deposition of A␤ in the brains of elderly subjects, in the absence of signs of neuronal degeneration and dementia (9 -11). The reasons for the absence of pathogenic effect exerted by A␤ in normal aging are unknown. The issue has important therapeutic implications, because the major strategies to prevent and cure AD are focused on halting A␤ accumulation (12).In brains from Alzheimer disease (AD) and Down syndrome patients, three major species of soluble A␤ have been identified by mass spectrometry: the full-length form, A␤1-42, which has a relative molecular mass of 4.5 kDa, and two N-terminal peptides truncated at residue 3 (A␤3-42) and residue 11 (A␤11-42) with relative molecular masses of 4.2 and 3.5 kDa, respectively (13, 14). The 4.2-and 3.5-kDa bands are more prominent in familial AD carrying presenilin 1 mutations than in sporadic AD, suggesting that the ratio of soluble A␤ species may dictate the toxicity of the aggregates (15).We predicted that the composition of soluble A␤ underlies the different effect exerted by the molecule in AD and in normal aging. To investigate this hypothesis, we studied the composition and properties of aggregation and toxicity as well as the damage produced on artificial membranes of soluble A␤, comparing these areas in sporadic AD and cognitively normal elderly subjects with abundant amyloid plaques in cerebral cortex. MATERIALS AND METHODSTissues-We used frozen blocks and formalin-fixed sections of frontal cortex from 14 cases with late onset sporadic AD (mean age at death 80 Ϯ ...

No abstract

Selenoprotein N (SEPN1) is a broadly expressed resident protein of the endoplasmic reticulum (ER) whose loss-of-function inexplicably leads to human muscle disease. We found that SEPN1 levels parallel those of endoplamic reticulum oxidoreductin 1 (ERO1), an ER protein thiol oxidase, and that SEPN1's redox activity defends the ER from ERO1-generated peroxides. Moreover, we have defined the redox-regulated interactome of SEPN1 and identified the ER calcium import SERCA2 pump as a redox-partner of SEPN1. SEPN1 enhances SERCA2 activity by reducing luminal cysteines that are hyperoxidized by ERO1-generated peroxides. Cells lacking SEPN1 are hypersensitive to ERO1 overexpression and conspicuously defective in ER calcium re-uptake. After being muscle-transduced with an adeno-associated virus driving ERO1α, SEPN1 knockout mice unmasks a myopathy that resembles the dense core disease due to human mutations in SEPN1, whereas the combined attenuation of ERO1α and SEPN1 enhances cell fitness. These observations reveal the involvement of SEPN1 in ER redox and calcium homeostasis and that an ERO1 inhibitor, restoring redox-dependent calcium homeostasis, may ameliorate the myopathy of SEPN1 deficiency.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of ␣v␤3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the ␣-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind ␣v␤3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind ␣v␤3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (␣v␤5, ␣v␤6, ␣v␤8, and ␣5␤1), although with 10 -100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas ␣-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.Various peptides containing the Asn-Gly-Arg (NGR) motif have been discovered by peptide-phage library panning in tumor-bearing mice (1). The tumor-homing properties of these peptides rely on the interaction with aminopeptidase N (CD13), a membrane protease expressed by the tumor neovasculature (2, 3). Because of this property, these peptides have been exploited for ligand-directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity (4). For instance, we have shown that peptides containing cyclic CNGRC and linear GNGRG motives can be used for delivering tumor necrosis factor ␣ (TNF) 3 (5-7), interferon ␥ (8 -10), and liposomal doxorubicin (11, 12) to tumor neovasculature, improving their therapeutic properties. The CNGRC-TNF conjugate, called NGR-TNF, is currently tested in phase II clinical studies (13-15). Other investigators have used the NGR motif embedded in similar or different molecular scaffolds for delivering chemotherapeutic drugs, antiangiogenic drugs, tissue factor, viruses, and other compounds to tumor vessels (1, 16 -32). Recently, a CNGRC peptide with an acetylated N-terminal ␣-amino group has been successfully exploited also for quantitative molecular magnetic resonance imaging of ...

Insufficient folding capacity of the endoplasmic reticulum (ER) activates the unfolded protein response (UPR) to restore homeostasis. Yet, how the UPR achieves ER homeostatic readjustment is poorly investigated, as in most studies the ER stress that is elicited cannot be overcome. Here we show that a proteostatic insult, provoked by persistent expression of the secretory heavy chain of immunoglobulin M (µs), is well-tolerated in HeLa cells. Upon µs expression, its levels temporarily eclipse those of the ER chaperone BiP, leading to acute, full-geared UPR activation. Once BiP is in excess again, the UPR transitions to chronic, submaximal activation, indicating that the UPR senses ER stress in a ratiometric fashion. In this process, the ER expands about three-fold and becomes dominated by BiP. As the UPR is essential for successful ER homeostatic readjustment in the HeLa-µs model, it provides an ideal system for dissecting the intricacies of how the UPR evaluates and alleviates ER stress.