PD-1, a T cell checkpoint receptor and target of cancer immunotherapy, is also expressed on myeloid cells. The role of myeloid-specific versus T cell–specific PD-1 ablation on antitumor immunity has remained unclear because most studies have used either PD-1–blocking antibodies or complete PD-1 KO mice. We generated a conditional allele, which allowed myeloid-specific (PD-1f/fLysMcre) or T cell–specific (PD-1f/fCD4cre) targeting of Pdcd1 gene. Compared with T cell–specific PD-1 ablation, myeloid cell–specific PD-1 ablation more effectively decreased tumor growth. We found that granulocyte/macrophage progenitors (GMPs), which accumulate during cancer-driven emergency myelopoiesis and give rise to myeloid-derived suppressor cells (MDSCs), express PD-1. In tumor-bearing PD-1f/fLysMcre but not PD-1f/fCD4cre mice, accumulation of GMP and MDSC was prevented, whereas systemic output of effector myeloid cells was increased. Myeloid cell–specific PD-1 ablation induced an increase of T effector memory cells with improved functionality and mediated antitumor protection despite preserved PD-1 expression in T cells. In PD-1–deficient myeloid progenitors, growth factors driving emergency myelopoiesis induced increased metabolic intermediates of glycolysis, pentose phosphate pathway, and TCA cycle but, most prominently, elevated cholesterol. Because cholesterol is required for differentiation of inflammatory macrophages and DC and promotes antigen-presenting function, our findings indicate that metabolic reprogramming of emergency myelopoiesis and differentiation of effector myeloid cells might be a key mechanism of antitumor immunity mediated by PD-1 blockade.

PDZK1, a multi-PDZ domain containing adaptor protein, interacts with various membrane proteins, including the high density lipoprotein (HDL) receptor scavenger receptor class B type I (SR-BI). Here we show that PDZK1 controls in a tissue-specific and post-transcriptional fashion the expression of SR-BI in vivo. SR-BI protein expression in PDZK1 knock-out (KO) mice was reduced by 95% in the liver, 50% in the proximal intestine, and not affected in steroidogenic organs (adrenal, ovary, and testis). Thus, PDZK1 joins a growing list of adaptors that control tissue-specific activity of cell surface receptors. Hepatic expression of SR-BII, a minor splice variant with an alternative C-terminal cytoplasmic domain, was not affected in PDZK1 KO mice, suggesting that binding of PDZK1 to SR-BI is required for controlling hepatic SR-BI expression. The loss of hepatic SR-BI was the likely cause of the elevation in plasma total and HDL cholesterol and the increase in HDL particle size in PDZK1 KO mice, phenotypes similar to those observed in SR-BI KO mice. PDZK1 KO mice differed from SR-BI KO mice in that the ratio of unesterified to total plasma cholesterol was normal, females were fertile, and cholesteryl ester stores in steroidogenic organs were essentially unaffected. These differences may be due to nearly normal extrahepatic expression of SR-BI in PDZK1 KO mice. The PDZK1-dependent regulation of hepatic SR-BI and, thus, lipoprotein metabolism supports the proposal that this adaptor may represent a new target for therapeutic intervention in cardiovascular disease.

The organization of lin genes and IS6100 was studied in three strains of Sphingomonas paucimobilis (B90A, Sp؉, and UT26) which degraded hexachlorocyclohexane (HCH) isomers but which had been isolated at different geographical locations. DNA-DNA hybridization data revealed that most of the lin genes in these strains were associated with IS6100, an insertion sequence classified in the IS6 family and initially found in Mycobacterium fortuitum. Eleven, six, and five copies of IS6100 were detected in B90A, Sp؉, and UT26, respectively. IS6100 elements in B90A were sequenced from five, one, and one regions of the genomes of B90A, Sp؉, and UT26, respectively, and were found to be identical. DNA-DNA hybridization and DNA sequencing of cosmid clones also revealed that S. paucimobilis B90A contains three and two copies of linX and linA, respectively, compared to only one copy of these genes in strains Sp؉ and UT26. Although the copy number and the sequence of the remaining genes of the HCH degradative pathway (linB, linC, linD, and linE) were nearly the same in all strains, there were striking differences in the organization of the linA genes as a result of replacement of portions of DNA sequences by IS6100, which gave them a strange mosaic configuration. Spontaneous deletion of linD and linE from B90A and of linA from Sp؉ occurred and was associated either with deletion of a copy of IS6100 or changes in IS6100 profiles. The evidence gathered in this study, coupled with the observation that the G؉C contents of the linA genes are lower than that of the remaining DNA sequence of S. paucimobilis, strongly suggests that all these strains acquired the linA gene through horizontal gene transfer mediated by IS6100. The association of IS6100 with the rest of the lin genes further suggests that IS6100 played a role in shaping the current lin gene organization.Hexachlorocyclohexane (HCH) was introduced for the control of agricultural pests and of vector-borne diseases in early 1940s. While this compound was used extensively all over the world, several reports on the persistence of HCH isomers (␣, ␤, ␥, and ␦) and their toxic effects on nontarget organisms appeared in the 1980s (11). These reports finally resulted in a ban on or restricted use of HCH in most countries. Neither the ban nor the restricted use has, however, reduced the levels of HCH residues in the environment (6, 25, 33), especially in soils that had a previous history of HCH application (2). One serious problem is the uptake of HCH residues from soil by crops, which then enter food products (1, 31). In addition to no further use of HCH, a decontamination program for HCHpolluted soils would diminish the risk posed by HCH residues to human, plant, and animal health. One possibility for decontamination is spontaneous or induced microbial degradation. Unfortunately, spontaneous microbial degradation of HCH isomers proceeds rather slowly (10, 15), although a number of bacteria which can degrade one or more isomers of HCH have been isolated. Thus, addition of naturally occu...

Proteins containing PDZ domains are involved in a large number of biological functions, including protein scaffolding, organization of ion channels, and signal transduction. We recently identified a novel PDZ domaincontaining protein, PDZK1, that is selectively expressed in normal tissues, where it is associated and colocalized with MAP17, a small 17-kDa membrane-associated protein; cMOAT, an organic anion transporter implicated in multidrug resistance; and the type IIa Na/Pi cotransporter. The protein cluster formed by PDZK1, MAP17, and cMOAT is upregulated in a significant number of human carcinomas originating in the colon, breast, lung, and kidney. In order to better define the function of PDZK1 in the protein cluster and its potential role in the organization of ion channels, we generated a PDZK1 knockout mouse. While PDZK1-deficient mice developed normally, did not display any gross phenotypic abnormalities, and were fecund, lack of PDZK1 resulted in modulation of expression of selective ion channels in the kidney, as well as increased serum cholesterol levels. However, no significant redistribution of proteins known to interact with PDZK1, such as MAP17, cMOAT, and the type IIa Na/Pi cotransporter, was observed. The absence of a more significant phenotype in PDZK1-deficient mice may be due to functional compensation by other PDZ domain-containing proteins, which could be instrumental in determining the location of interacting proteins such as ion channels and other membrane-associated proteins in defined areas of the plasma membrane. PDZK1, a recently described protein containing four PDZ domains, belongs to a cluster of proteins including MAP17 and cMOAT (7-10). All three proteins are upregulated in human carcinomas arising in the kidney, lung, colon, and breast. Although the exact function of PDZK1 is unknown, it has been postulated that it plays a role in multidrug resistance through its interaction with the organic anion transporter cMOAT, also known as MRP2, the multidrug resistance-associated protein (9,11,12,14,24). More recently, PDZK1 has been found to interact with the type IIa Na/Pi cotransporter and therefore may participate in the apical sorting of ion channels (4). Furthermore, PDZK1 is overexpressed in estrogen receptor-positive breast carcinomas compared to estrogen-negative tumors, suggesting a role for PDZK1 in tissue response to ␤-estradiol (3). PDZ domains were originally recognized as structural motifs in the mammalian postsynaptic density protein PSD-95 (1), the Drosophila disk large tumor suppressor Dlg (26), and the tight junction protein ZO-1 (25). Such domains, typically 80 to 120 amino acids, bind to well-defined consensus sequences and have been described in a number of proteins associated with specialized areas of the plasma membrane (2, 22). PDZ domain-containing proteins are involved in synaptic organization, control of cell proliferation, and cell differentiation (1,6,13,20,26). Some of these proteins contain several PDZ domains and, as a result, promote the clustering of a...

No abstract

PDZK1 is a four-PDZ domain-containing scaffold protein that, via its first PDZ domain (PDZ1), binds to the C terminus of the high density lipoprotein (HDL) receptor scavenger receptor, class B, type I (SR-BI). Abolishing PDZK1 expression in PDZK1 knock-out (KO) mice leads to a post-transcriptional, tissue-specific decrease in SR-BI protein level and an increase in total plasma cholesterol carried in abnormally large HDL particles. Here we show that, although hepatic overexpression of PDZK1 restored normal SR-BI protein abundance and function in PDZK1 KO mice, hepatic overexpression of only the PDZ1 domain was not sufficient to restore normal SR-BI function. In wild-type mice, overexpression of the PDZ1 domain overcame the activity of the endogenous hepatic PDZK1, resulting in a 75% reduction in hepatic SR-BI protein levels and intracellular mislocalization of the remaining SR-BI. As a consequence, the plasma lipoproteins in PDZ1 transgenic mice resembled those in PDZK1 KO mice (hypercholesterolemia due to large HDL). These results indicate that the PDZ1 domain can control the abundance and localization, and therefore the function, of hepatic SR-BI and that structural features of PDZK1 in addition to its SR-BI-binding PDZ1 domain are required for normal hepatic SR-BI regulation.The high density lipoprotein (HDL) 3 receptor SR-BI (scavenger receptor, class B, type I) plays a key role in lipoprotein metabolism (1) by mediating the cellular uptake of cholesteryl esters from HDL and other lipoproteins into cells (2-7) via a mechanism called selective lipid uptake (3, 8 -10). SR-BI also mediates bidirectional flux of unesterified cholesterol between cells and lipoproteins (11)(12)(13)(14).Most in vivo analyses of the physiological function of SR-BI have been conducted in mice. SR-BI is highly expressed in the liver and steroidogenic tissues (adrenal gland, ovary, and testis), which exhibit the highest levels of HDL cholesterol uptake (3). Experimental manipulations of murine SR-BI expression (e.g. hepatic overexpression, homozygous null gene knock-out) can lead to changes in total plasma cholesterol levels, the ratio of plasma unesterified to total cholesterol, HDL structure, biliary cholesterol secretion, the amounts of cholesterol stored in steroidogenic tissues, and susceptibility to atherosclerosis (15-27). Homozygous null SR-BI knock-out mice exhibit abnormally elevated (ϳ2.2-fold) plasma cholesterol in large HDL particles with a unesterified cholesterol/total cholesterol ratio roughly double that of wild-type (WT) mice (19,52). This dyslipidemia is associated with female infertility and defects in the maturation and/or structure as well as reductions in the survival times of red blood cells (21,28,29,30).To date, only one intracellular protein has been shown to interact directly with and influence the function of SR-BI (31). This protein, PDZK1, regulates SR-BI expression in a tissuespecific, post-transcriptional manner (32). PDZK1 is a scaffold protein containing four PDZ protein interaction domains (Fig. 1A...

PDZK1 is a scaffold protein containing four PDZ protein interaction domains, which bind to the carboxy termini of a number of membrane transporter proteins, including ion channels (e.g., CFTR) and cell surface receptors. One of these, the HDL receptor, scavenger receptor class B type I (SR-BI), exhibits a striking, tissue-specific dependence on PDZK1 for its expression and activity. In PDZK1 knockout (KO) mice there is a marked reduction of SR-BI protein expression (approximately 95%) in the liver, but not in steroidogenic tissues or, as we show in this report, in bone marrow- or spleen-derived macrophages, or lung-derived endothelial cells. Because of hepatic SR-BI deficiency, PDZK1 KO mice exhibit dyslipidemia characterized by elevated plasma cholesterol carried in abnormally large HDL particles. Here, we show that inactivation of the PDZK1 gene promotes the development of aortic root atherosclerosis in apolipoprotein E (apoE) KO mice fed with a high fat/high cholesterol diet. However, unlike complete SR-BI-deficiency in SR-BI/apoE double KO mice, PDZK1 deficiency in PDZK1/apoE double knockout mice did not result in development of occlusive coronary artery disease or myocardial infarction, presumably because of their residual expression of SR-BI. These findings demonstrate that deficiency of an adaptor protein essential for normal expression of a lipoprotein receptor promotes atherosclerosis in a murine model. They also define PDZK1 as a member of the family of proteins that is instrumental in preventing cardiovascular disease by maintaining normal lipoprotein metabolism.

PDZK1 is a multi-PDZ domain-containing adaptor protein that binds to the C terminus of the high density lipoprotein receptor, scavenger receptor, class B, type I (SR-BI), and controls the posttranscriptional, tissue-specific expression of this lipoprotein receptor. In the absence of PDZK1 (PDZK1(؊/؊) mice), murine hepatic SR-BI protein levels are very low (<5% of control). As a consequence, abnormal plasma lipoprotein metabolism (ϳ1.5-1.7-fold increased total plasma cholesterol carried in both normal size and abnormally large high density lipoprotein particles) resembles, but is not as severely defective The HDL 2 receptor SR-BI plays an important role in lipoprotein-mediated lipid transport and metabolism (1). SR-BI mediates HDL binding to cells and subsequently facilitates the net transfer of cholesteryl esters from the particle core but not the protein or most of the lipid components of the outer shell of the lipoprotein, a process called selective lipid uptake (1-4). This receptor also mediates bidirectional movement of unesterified cholesterol between cells and lipoproteins (5-7). SR-BI is most highly expressed in hepatocytes, where it helps control plasma lipoprotein metabolism, and in steroidogenic cells, where it delivers lipoprotein cholesterol for storage and subsequent conversion into steroid hormones (4,8,9). Normal expression and moderate transgene-mediated overexpression of hepatic SR-BI have been shown to protect against atherosclerosis in several murine models (10 -16), and SR-BI transgene expression in the liver can prevent the female infertility seen in otherwise SR-BI null mice (SR-BI (Ϫ/Ϫ)) (12,17,18).SR-BI deficiency in SR-BI (Ϫ/Ϫ) mice causes an ϳ2-fold elevation in plasma cholesterol carried in both normal size and abnormally large HDL particles (12, 19), which exhibit an abnormally high ratio of unesterified cholesterol-to-total (unesterified plus esterified) cholesterol (14, 20) as well as a 30 -50% decrease in biliary cholesterol secretion rates and concentrations (12,21,22). These phenotypes are thought to be consequences of the reduced hepatic uptake of cholesterol from circulating HDL and can be reversed by hepatic SR-BI transgene expression (17,21,23,24).Tissue SR-BI expression can be regulated by both transcriptional and posttranscriptional mechanisms (25). In the liver, but not in steroidogenic tissues, posttranscriptional control of SR-BI protein expression depends on the presence of an adaptor protein, PDZK1 (26 -28). Cytoplasmic adaptor proteins that bind to membrane-associated proteins regulate a variety of biological processes, including signal transduction, adhesion, membrane trafficking, and cellular transport (29). They often comprise combinations of modular protein interaction domains such as Src homology (SH2, SH3), phosphotyrosinebinding (PTB), and PDZ domains that recognize short peptide or phosphopeptide motifs (e.g. PDZ domains usually bind to the C-terminal 3-4 residues of interacting proteins) (30).PDZK1 is a single chain, four-PDZ domain-containing polypeptide tha...