Functional characterization of the disease-associated CCL2 rs1024611G-rs13900T haplotype: The role of the RNA-binding protein HuR.

CC-chemokine ligand 2 (CCL2) is involved in the pathogenesis of several diseases associated with monocyte/macrophage recruitment, such as HIV-associated neurocognitive disorder (HAND), tuberculosis, and atherosclerosis. The rs1024611 (alleles:A>G; G is the risk allele) polymorphism in the CCL2 cis-regulatory region is associated with increased CCL2 expression in vitro and ex vivo, leukocyte mobilization in vivo, and deleterious disease outcomes. However, the molecular basis for the rs1024611-associated differential CCL2 expression remains poorly characterized. It is conceivable that genetic variant(s) in linkage disequilibrium (LD) with rs1024611 could mediate such effects. Previously, we used rs13900 (alleles:_C>T) in the CCL2 3' untranslated region (3' UTR) that is in perfect LD with rs1024611 to demonstrate allelic expression imbalance (AEI) of CCL2 in heterozygous individuals. Here we tested the hypothesis that the rs13900 could modulate CCL2 expression by altering mRNA turnover and/or translatability. The rs13900 T allele conferred greater stability to the CCL2 transcript when compared to the rs13900 C allele. The rs13900 T allele also had increased binding to Human Antigen R (HuR), an RNA-binding protein, in vitro and ex vivo. The rs13900 alleles imparted differential activity to reporter vectors and influenced the translatability of the reporter transcript. We further demonstrated a role for HuR in mediating allele-specific effects on CCL2 expression in overexpression and silencing studies. The presence of the rs1024611G-rs13900T conferred a distinct transcriptomic signature related to inflammation and immunity. Our studies suggest that the differential interactions of HuR with rs13900 could modulate CCL2 expression and explain the interindividual differences in CCL2-mediated disease susceptibility.

Association of HIV-1 Infection and Antiretroviral Therapy With Type 2 Diabetes in the Hispanic Population of the Rio Grande Valley, Texas, USA.

The Rio Grande Valley (RGV) in South Texas has one of the highest prevalence of obesity and type 2 diabetes (T2D) in the United States (US). We report for the first time the T2D prevalence in persons with HIV (PWH) in the RGV and the interrelationship between T2D, cardiometabolic risk factors, HIV-related indices, and antiretroviral therapies (ART). The PWH in this study received medical care at Valley AIDS Council (VAC) clinic sites located in Harlingen and McAllen, Texas. Henceforth, this cohort will be referred to as Valley AIDS Council Cohort (VACC). Cross-sectional analyses were conducted using retrospective data obtained from 1,827 registries. It included demographic and anthropometric variables, cardiometabolic traits, and HIV-related virological and immunological indices. For descriptive statistics, we used mean values of the quantitative variables from unbalanced visits across 20 months. Robust regression methods were used to determine the associations. For comparisons, we used cardiometabolic trait data obtained from HIV-uninfected San Antonio Mexican American Family Studies (SAMAFS; N = 2,498), and the Mexican American population in the National Health and Nutrition Examination Survey (HHANES; N = 5,989). The prevalence of T2D in VACC was 51% compared to 27% in SAMAFS and 19% in HHANES, respectively. The PWH with T2D in VACC were younger (4.7 years) and had lower BMI (BMI 2.43 units less) when compared to SAMAFS individuals. In contrast, VACC individuals had increased blood pressure and dyslipidemia. The increased T2D prevalence in VACC was independent of BMI. Within the VACC, ART was associated with viral load and CD4+ T cell counts but not with metabolic dysfunction. Notably, we found that individuals with any INSTI combination had higher T2D risk: OR 2.08 (95%CI 1.67, 2.6; p < 0.001). In summary, our results suggest that VACC individuals may develop T2D at younger ages independent of obesity. The high burden of T2D in these individuals necessitates rigorously designed longitudinal studies to draw potential causal inferences and develop better treatment regimens.

Further evidence supporting a potential role for ADH1B in obesity.

Insulin is an essential hormone that regulates glucose homeostasis and metabolism. Insulin resistance (IR) arises when tissues fail to respond to insulin, and it leads to serious health problems including Type 2 Diabetes (T2D). Obesity is a major contributor to the development of IR and T2D. We previously showed that gene expression of alcohol dehydrogenase 1B (ADH1B) was inversely correlated with obesity and IR in subcutaneous adipose tissue of Mexican Americans. In the current study, a meta-analysis of the relationship between ADH1B expression and BMI in Mexican Americans, African Americans, Europeans, and Pima Indians verified that BMI was increased with decreased ADH1B expression. Using established human subcutaneous pre-adipocyte cell lines derived from lean (BMI < 30 kg m-2) or obese (BMI ≥ 30 kg m-2) donors, we found that ADH1B protein expression increased substantially during differentiation, and overexpression of ADH1B inhibited fatty acid binding protein expression. Mature adipocytes from lean donors expressed ADH1B at higher levels than obese donors. Insulin further induced ADH1B protein expression as well as enzyme activity. Knockdown of ADH1B expression decreased insulin-stimulated glucose uptake. Our findings suggest that ADH1B is involved in the proper development and metabolic activity of adipose tissues and this function is suppressed by obesity.

Overexpression of TC-PTP in murine epidermis attenuates skin tumor formation.

T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, has been shown to function as a tumor suppressor during skin carcinogenesis. In the current study, we generated a novel epidermal-specific TC-PTP-overexpressing (K5HA.Ptpn2) mouse model to show that TC-PTP contributes to the attenuation of chemically induced skin carcinogenesis through the synergistic regulation of STAT1, STAT3, STAT5, and PI3K/AKT signaling. We found overexpression of TC-PTP increased epidermal sensitivity to DMBA-induced apoptosis and it decreased TPA-mediated hyperproliferation, coinciding with reduced epidermal thickness. Inhibition of STAT1, STAT3, STAT5, or AKT reversed the effects of TC-PTP overexpression on epidermal survival and proliferation. Mice overexpressing TC-PTP in the epidermis developed significantly reduced numbers of tumors during skin carcinogenesis and presented a prolonged latency of tumor initiation. Examination of human papillomas and squamous cell carcinomas (SCCs) revealed that TC-PTP expression was significantly reduced and TC-PTP expression was inversely correlated with the increased grade of SCCs. Our findings demonstrate that TC-PTP is a potential therapeutic target for the prevention of human skin cancer given that it is a major negative regulator of oncogenic signaling.

The role of T-cell protein tyrosine phosphatase in epithelial carcinogenesis.

T-cell protein tyrosine phosphatase (TC-PTP, encoded by PTPN2) is a nonreceptor PTP that is most highly expressed in hematopoietic tissues. TC-PTP modulates a variety of physiological functions including cell cycle progression, cell survival and proliferation, and hematopoiesis through tyrosine dephosphorylation of its target substrates, such as EGFR, JAK1, JAK3, STAT1, and STAT3. Studies with whole or tissue-specific loss of TC-PTP function transgenic mice have shown that TC-PTP has crucial roles in the regulation of the immune response, insulin signaling, and oncogenic signaling. More recently, the generation of epidermal-specific TC-PTP-deficient mice for use in multistage skin carcinogenesis bioassays demonstrated that TC-PTP suppresses skin tumor formation by negatively regulating STAT3 and AKT signaling. Further investigation showed that TC-PTP also minimizes UVB-induced epidermal cell damage by promoting apoptosis through the negative regulation of Flk-1/JNK signaling. These findings provide major evidence for a tumor suppressive function for TC-PTP against environment-induced skin cancer. Here, we will discuss TC-PTP, its substrates, and its functions with an emphasis on its role in skin carcinogenesis.

Protein Tyrosine Phosphatases as Potential Regulators of STAT3 Signaling.

The signal transducer and activator of transcription 3 (STAT3) protein is a major transcription factor involved in many cellular processes, such as cell growth and proliferation, differentiation, migration, and cell death or cell apoptosis. It is activated in response to a variety of extracellular stimuli including cytokines and growth factors. The aberrant activation of STAT3 contributes to several human diseases, particularly cancer. Consequently, STAT3-mediated signaling continues to be extensively studied in order to identify potential targets for the development of new and more effective clinical therapeutics. STAT3 activation can be regulated, either positively or negatively, by different posttranslational mechanisms including serine or tyrosine phosphorylation/dephosphorylation, acetylation, or demethylation. One of the major mechanisms that negatively regulates STAT3 activation is dephosphorylation of the tyrosine residue essential for its activation by protein tyrosine phosphatases (PTPs). There are seven PTPs that have been shown to dephosphorylate STAT3 and, thereby, regulate STAT3 signaling: PTP receptor-type D (PTPRD), PTP receptor-type T (PTPRT), PTP receptor-type K (PTPRK), Src homology region 2 (SH-2) domain-containing phosphatase 1(SHP1), SH-2 domain-containing phosphatase 2 (SHP2), MEG2/PTP non-receptor type 9 (PTPN9), and T-cell PTP (TC-PTP)/PTP non-receptor type 2 (PTPN2). These regulators have great potential as targets for the development of more effective therapies against human disease, including cancer.

Epidermal-specific deletion of TC-PTP promotes UVB-induced epidermal cell survival through the regulation of Flk-1/JNK signaling.

UVB exposure can contribute to the development of skin cancer by modulating protein tyrosine kinase (PTK) signaling. It has been suggested that UVB radiation increases the ligand-dependent activation of PTKs and induces PTP inactivation. Our recent studies have shown that T-cell protein tyrosine phosphatase (TC-PTP) attenuates skin carcinogenesis induced by chemical regimens, which indicates its critical role in the prevention of skin cancer. In the current work, we report that TC-PTP increases keratinocyte susceptibility to UVB-induced apoptosis via the downregulation of Flk-1/JNK signaling. We showed that loss of TC-PTP led to resistance to UVB-induced apoptosis in vivo epidermis. We established immortalized primary keratinocytes (IPKs) from epidermal-specific TC-PTP-deficient (K14Cre.Ptpn2fl/fl) mice. Immortalized TC-PTP-deficient keratinocytes (TC-PTP/KO IPKs) showed increased cell survival against UVB-induced apoptosis which was concomitant with a UVB-mediated increase in Flk-1 phosphorylation, especially on tyrosine residue 1173. Inhibition of Flk-1 by either its specific inhibitors or siRNA in TC-PTP/KO IPKs reversed this effect and significantly increased cell death after UVB irradiation in comparison with untreated TC-PTP/KO IPKs. Immunoprecipitation analysis using the TC-PTP substrate-trapping mutant TCPTP-D182A indicated that TC-PTP directly interacts with Flk-1 to dephosphorylate it and their interaction was stimulated by UVB. Following UVB-mediated Flk-1 activation, the level of JNK phosphorylation was also significantly increased in TC-PTP/KO IPKs compared to control IPKs. Similar to our results with Flk-1, treatment of TC-PTP/KO IPKs with the JNK inhibitor SP600125 significantly increased apoptosis after UVB irradiation, confirming that the effect of TC-PTP on UVB-mediated apoptosis is regulated by Flk-1/JNK signaling. Western blot analysis showed that both phosphorylated Flk-1 and phosphorylated JNK were significantly increased in the epidermis of TC-PTP-deficient mice compared to control mice following UVB. Our results suggest that TC-PTP plays a protective role against UVB-induced keratinocyte cell damage by promoting apoptosis via negative regulation of Flk-1/JNK survival signaling.

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation.

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation.

Targeted disruption of TC-PTP in the proliferative compartment augments STAT3 and AKT signaling and skin tumor development.

Tyrosine phosphorylation is a vital mechanism that contributes to skin carcinogenesis. It is regulated by the counter-activities of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Here, we report the critical role of T-cell protein tyrosine phosphatase (TC-PTP), encoded by Ptpn2, in chemically-induced skin carcinogenesis via the negative regulation of STAT3 and AKT signaling. Using epidermal specific TC-PTP knockout (K14Cre.Ptpn2fl/fl) mice, we demonstrate loss of TC-PTP led to a desensitization to tumor initiator 7,12-dimethylbenz[a]anthracene (DMBA)-induced apoptosis both in vivo epidermis and in vitro keratinocytes. TC-PTP deficiency also resulted in a significant increase in epidermal thickness and hyperproliferation following exposure to the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis showed that both phosphorylated STAT3 and phosphorylated AKT expressions were significantly increased in epidermis of TC-PTP-deficient mice compared to control mice following TPA treatment. Inhibition of STAT3 or AKT reversed the effects of TC-PTP deficiency on apoptosis and proliferation. Finally, TC-PTP knockout mice showed a shortened latency of tumorigenesis and significantly increased numbers of tumors during two-stage skin carcinogenesis. Our findings reveal that TC-PTP has potential as a novel target for the prevention of skin cancer through its role in the regulation of STAT3 and AKT signaling.

GFRA1 promotes cisplatin-induced chemoresistance in osteosarcoma by inducing autophagy.

Recent progress in chemotherapy has significantly increased its efficacy, yet the development of chemoresistance remains a major drawback. In this study, we show that GFRA1/GFRα1 (GDNF family receptor α 1), contributes to cisplatin-induced chemoresistance by regulating autophagy in osteosarcoma. We demonstrate that cisplatin treatment induced GFRA1 expression in human osteosarcoma cells. Induction of GFRA1 expression reduced cisplatin-induced apoptotic cell death and it significantly increased osteosarcoma cell survival via autophagy. GFRA1 regulates AMPK-dependent autophagy by promoting SRC phosphorylation independent of proto-oncogene RET kinase. Cisplatin-resistant osteosarcoma cells showed NFKB1/NFκB-mediated GFRA1 expression. GFRA1 expression promoted tumor formation and growth in mouse xenograft models and inhibition of autophagy in a GFRA1-expressing xenograft mouse model during cisplatin treatment effectively reduced tumor growth and increased survival. In cisplatin-treated patients, treatment period and metastatic status were associated with GFRA1-mediated autophagy. These findings suggest that GFRA1-mediated autophagy is a promising novel target for overcoming cisplatin resistance in osteosarcoma.

Activation of T-cell protein-tyrosine phosphatase suppresses keratinocyte survival and proliferation following UVB irradiation.

Chronic exposure to UV radiation can contribute to the development of skin cancer by promoting protein-tyrosine kinase (PTK) signaling. Studies show that exposure to UV radiation increases the ligand-independent activation of PTKs and induces protein-tyrosine phosphatase (PTP) inactivation. In the present work, we report that T-cell PTP (TC-PTP) activity is stimulated during the initial response to UVB irradiation, which leads to suppression of keratinocyte cell survival and proliferation via the down-regulation of STAT3 signaling. Our results show that TC-PTP-deficient keratinocyte cell lines expressed a significantly increased level of phosphorylated STAT3 after exposure to low dose UVB. This increase corresponded with increased cell proliferation in TC-PTP-deficient keratinocytes following UVB irradiation. Loss of TC-PTP also reduced UVB-induced apoptosis. Corroborating with these results, overexpression of TC-PTP in keratinocyte cell lines yielded a decrease in phosphorylated STAT3 levels, which corresponded with a significant decrease in cell proliferation in response to low dose UVB. We demonstrate that TC-PTP activity was increased upon UVB exposure, and overexpression of TC-PTP in keratinocyte cell lines further increased its activity in the presence of UVB. Treatment of TC-PTP-deficient keratinocytes with the STAT3 inhibitor STA21 significantly reduced cell viability following UVB exposure in comparison with untreated TC-PTP-deficient keratinocytes, confirming that the effect of TC-PTP on cell viability is mediated by STAT3 dephosphorylation. Combined, our results indicate that UVB-mediated activation of TC-PTP plays an important role in the STAT3-dependent regulation of keratinocyte cell proliferation and survival. Furthermore, these results suggest that TC-PTP may be a novel potential target for the prevention of UVB-induced skin cancer.

Protein tyrosine phosphatases PTP-1B, SHP-2, and PTEN facilitate Rb/E2F-associated apoptotic signaling.

To maintain tissue homeostasis, apoptosis is functionally linked to the cell cycle through the retinoblastoma (Rb)/E2F pathway. When the Rb tumor suppressor protein is functionally inactivated, E2F1 elicits an apoptotic response through both intrinsic (caspase-9 mediated) and extrinsic (caspase-8 mediated) apoptotic pathways in order to eliminate hyperproliferative cells. Rb/E2F-associated apoptosis has been demonstrated to be associated with the loss of constitutive transcriptional repression by Rb/E2F complexes and mediated by caspase-8. Protein tyrosine phosphatases (PTPs) PTP-1B and SHP-2 have been previously shown to be directly activated by loss of Rb/E2F repression during Rb/E2F-associated apoptosis. In this current study, we demonstrate that the PTEN tumor suppressor is also directly activated by loss of Rb/E2F repression. We also demonstrate that PTP-1B, SHP-2, and PTEN play a functional role in Rb/E2F-associated apoptosis. Knockdown of PTP1B, SHP2, or PTEN expression with small interfering RNA (siRNA) in apoptotic cells increases cell viability and rescues cells from the Rb/E2F-associated apoptotic response. Furthermore, rescue from apoptosis coincides with inhibition of caspase-8 and caspase-3 cleavage (activation). Our results indicate PTP-1B, SHP-2, and PTEN all play a functional role in Rb/E2F-associated apoptotic signal transduction and provide further evidence that PTP-1B, SHP-2, and PTEN can contribute to tumor suppression through an Rb/E2F-associated mechanism.

A green approach toward quinoxalines and bis-quinoxalines and their biological evaluation against A431, human skin cancer cell lines.

BACKGROUND: The objective of this study was to develop a practical green procedure to synthesize quinoxalines and bis-quinoxalines and evaluate their inhibitory effects on the viability of A431 human epidermoid carcinoma cells. METHOD: A series of quinoxaline and bis-quinoxaline derivatives have been designed and synthesized following a microwave-assisted and bismuth nitrate-catalyzed eco-friendly route. A detailed comparison has been made between microwave-induced protocol with the reactions occurred at room temperature. The structure of the compounds have been elucidated by various spectroscopic methods and finally confirmed by x-ray crystallographic analyses. RESULTS: Two quinoxaline derivatives, compounds 6 and 12 have demonstrated inhibitory effects on the viability of A431 human epidermoid carcinoma cells when compared with HaCaT nontumorigenic human keratinocyte cells. CONCLUSION: Notably, compound 6 inhibits Stat3 phosphorylation/activation in A431 skin cancer cells.

SHP-2 and PTP-pest induction during Rb-E2F associated apoptosis.

Apoptosis is intimately connected to cell cycle regulation via the Retinoblastoma (Rb)-E2F pathway and thereby serves an essential role in tumor suppression by eliminating aberrant hyperproliferative cells. Upon loss of Rb activity, an apoptotic response can be elicited through both p53-dependent and p53-independent mechanisms. While much of this apoptotic response has been attributed to the p19ARF/p53 pathway, increasing evidence has supported the role of protein tyrosine phosphatases (PTPs) in contributing to the initiation of the Rb-E2F-associated apoptotic response. One protein tyrosine phosphatase, PTP-1B, which is induced by the Rb-E2F pathway, has been shown to contribute to a p53-independent apoptotic pathway by inactivating focal adhesion kinase. This report identifies two additional PTPs, SHP-2 and PTP-PEST, that are also directly activated by the Rb-E2F pathway and which can contribute to signal transduction during p53-independent apoptosis.

Insulin-like growth factor-I mediates neuroprotection in proteasome inhibition-induced cytotoxicity in SH-SY5Y cells.

The proteasome is an enzyme complex responsible for targeted intracellular proteolysis. Alterations in proteasome-mediated protein clearance have been implicated in the pathogenesis of aging, Alzheimer's disease (AD) and Parkinson's disease (PD). In such diseases, proteasome inhibition may contribute to formation of abnormal protein aggregates, which in turn activate intracellular unfolded protein responses that cause oxidative stress and apoptosis. In this study, we investigated the protective effect of Insulin-like Growth Factor-I (IGF-1) for neural SH-SY5Y cells treated with the proteasomal inhibitor, Epoxomicin. In SH-SY5Y cells, Epoxomicin treatment results in accumulation of intracellular ubiquitinated proteins and cytochrome c release from damaged mitochondria, leading to cell death, in Epoxomicin time- and dose-dependent manner. In cells treated with small amounts of IGF-1, the same dosages of Epoxomicin reduced both mitochondrial damage (cytochrome c release) and reduced caspase-3 activation and PARP cleavage, both of which are markers of apoptosis. Notably, however, IGF-1-treated SH-SY5Y cells still contained ubiquitinated protein aggregates. This result indicates that IGF-1 blocks the downstream apoptotic consequences of Epoxomicin treatment leading to decreased proteasome function. Clues as to the mechanism for this protective effect come from (a) increased AKT phosphorylation observed in IGF-1-protected cells, vs. cells exposed to Epoxomicin without IGF-1, and (b) reduction of IGF-1 protection by pretreatment of the cells with LY294002 (an inhibitor of PI3-kinase). Together these findings suggest that activation of PI3/AKT pathways by IGF-1 is involved in IGF-1 neuroprotection against apoptosis following proteasome inhibition.

Stress-induced beta-lactam antibiotic resistance mutation and sequences of stationary-phase mutations in the Escherichia coli chromosome.

In some enterobacterial pathogens, but not in Escherichia coli, loss-of-function mutations are a common route to clinically relevant beta-lactam antibiotic resistance. We previously constructed an assay system for studying enterobacterial beta-lactam resistance mutations using the well-developed genetics of E. coli by integrating enterobacterial ampRC genes into the E. coli chromosome. Like the cells of other enterobacteria, E. coli cells acquire beta-lactam resistance by ampD mutation. Here we show that starvation and stress responses provoke ampD beta-lactam resistance mutagenesis. When starved on lactose medium, Lac(-) strains used in mutagenesis studies accumulate ampD beta-lactam resistance mutations independent of Lac reversion. DNA double-strand break repair (DSBR) proteins and the SOS and RpoS stress responses are required for this mutagenesis, in agreement with the results obtained for lac reversion in these cells. Surprisingly, the stress-induced ampD mutations require DinB (DNA polymerase IV) and partially require error-prone DNA polymerase V, unlike lac mutagenesis, which requires only DinB. This assay demonstrates that real-world stressors, such as starvation, can induce clinically relevant resistance mutations. Finally, we used the ampD system to observe the true forward-mutation sequence spectrum of DSBR-associated stress-induced mutagenesis, for which previously only frameshift reversions were studied. We found that base substitutions outnumber frameshift mutations, as seen in other experimental systems showing stress-induced mutagenesis. The important evolutionary implication is that not only loss-of-function mutations but also change-of-function mutations can be generated by this mechanism.

Single-strand-specific exonucleases prevent frameshift mutagenesis by suppressing SOS induction and the action of DinB/DNA polymerase IV in growing cells.

Escherichia coli strains carrying null alleles of genes encoding single-strand-specific exonucleases ExoI and ExoVII display elevated frameshift mutation rates but not base substitution mutation rates. We characterized increased spontaneous frameshift mutation in ExoI- ExoVII- cells and report that some of this effect requires RecA, an inducible SOS DNA damage response, and the low-fidelity, SOS-induced DNA polymerase DinB/PolIV, which makes frameshift mutations preferentially. We also find that SOS is induced in ExoI- ExoVII- cells. The data imply a role for the single-stranded exonucleases in guarding the genome against mutagenesis by removing excess single-stranded DNA that, if left, leads to SOS induction and PolIV-dependent mutagenesis. Previous results implicated PolIV in E. coli mutagenesis specifically during starvation or antibiotic stresses. Our data imply that PolIV can also promote mutation in growing cells under genome stress due to excess single-stranded DNA.

The platelet glycoprotein Ib-von Willebrand factor interaction activates the collagen receptor alpha2beta1 to bind collagen: activation-dependent conformational change of the alpha2-I domain.

Integrin alpha2beta1 (glycoprotein [GP] Ia/IIa) is a major platelet receptor for collagen, containing its collagen-binding site within the alpha2 I domain. alpha2beta1 changes conformation upon platelet activation, increasing its affinity for collagen. We observed that 2 antibodies known to bind within the alpha2I domain, 12F1 and 6F1, bound preferentially to adenosine diphosphate (ADP)-activated platelets. Interestingly, when whole blood was perfused over a surface coated with either 12F1 or 6F1, only 6F1 supported the adhesion of unstimulated platelets. To test whether the interaction of GP Ib with von Willebrand factor (VWF) directly activates alpha2beta1, we used 12F1 as a probe of integrin activation. We perfused blood over a surface coated with a mixture of VWF-A1 domain (a GP Ib ligand) and 12F1 or VWF-A1 and mouse immunoglobulin G (IgG). Platelets rolled and did not attach stably on the A1/IgG surface, but they firmly bound and covered the A1/12F1 surface. We corroborated that 12F1 binds an active conformation of the I domain by showing that it binds with higher affinity to a gain-of-function mutant than to either wild-type I domain or a loss-of-function mutant. These results strongly suggest that the interaction of platelet GP Ib with VWF mediates the activation of alpha2beta1, increasing its affinity for collagen.