Artificial antigen-presenting constructs efficiently stimulate minor histocompatibility antigen-specific cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) specific for hematopoietic-restricted minor histocompatibility antigens (mHags) are important reagents for adoptive immunotherapy of relapsed leukemia after allogeneic stem cell transplantation. However, expansion of these CTLs to therapeutic numbers is often hampered by the limited supply of antigen-presenting cells (APCs). Therefore, we evaluated whether cell-sized latex beads coated with HLA/mHag complexes HLA-A2/HA-1 or HLA-A2/HA-2 and recombinant CD80 and CD54 molecules can replace professional APCs. The artificial antigen-presenting constructs (aAPCs) effectively stimulated HA-1– and HA-2–specific CTL clones as shown by ligand-specific expansion, cytokine production, and maintenance of cytotoxic activity, without alteration of CTL phenotype. Furthermore, HA-1–specific polyclonal CTL lines were enriched as efficiently by aAPCs as by autologous HA-1 peptide-pulsed dendritic cells. Thus, aAPCs coated with HLA/mHag complexes, CD80, and CD54 may serve as tools for in vitro enrichment of immunotherapeutic mHag-specific CTL lines.      

The reactivity of Bw4+ HLA-B and HLA-A alleles with KIR3DL1: implications for patient and donor suitability for haploidentical stem cell transplantations

Natural killer (NK)–cell alloreactivity can be exploited in haploidentical hematopoietic stem cell transplantation (HSCT). NK cells from donors whose HLA type includes Bw4, a public epitope present on a subset of HLA-B alleles, can be alloreactive toward recipients whose cells lack Bw4. Serologically detectable epitopes related to Bw4 also exist on a subset of HLA-A alleles, but the interaction of these alleles with KIR3DL1 is controversial. We therefore undertook a systematic analysis of the ability of most common HLA-B alleles and HLA-A alleles with Bw4 serologic reactivity to protect target cells from lysis by KIR3DL1-dependent NK cells. All Bw4^– HLA-B alleles failed to protect target cells from lysis. All Bw4⁺ HLA-B alleles with the exception of HLA-B*1301 and -B*1302 protected targets from lysis. HLA-A*2402 and HLA-A*3201 unequivocally protected target cells from lysis, whereas HLA-A*2501 and HLA-A*2301 provided only weak protection from lysis. KIR3DL1-dependent alloreactive NK clones were identified in donors with HLA-A*2402 but not in donors with HLA-B*1301 or -B*1302. These findings clarify the HLA types that donors and recipients need in haploidentical HSCT and other NK allotherapies in order to benefit from NK alloreactivity.     

Crystal structure of the proteasomal deubiquitylation module Rpn8-Rpn11

The ATP-dependent degradation of polyubiquitylated proteins by the 26S proteasome is essential for the maintenance of proteome stability and the regulation of a plethora of cellular processes. Degradation of substrates is preceded by the removal of polyubiquitin moieties through the isopeptidase activity of the subunit Rpn11. Here we describe three crystal structures of the heterodimer of the Mpr1–Pad1–N-terminal domains of Rpn8 and Rpn11, crystallized as a fusion protein in complex with a nanobody. This fusion protein exhibits modest deubiquitylation activity toward a model substrate. Full activation requires incorporation of Rpn11 into the 26S proteasome and is dependent on ATP hydrolysis, suggesting that substrate processing and polyubiquitin removal are coupled. Based on our structures, we propose that premature activation is prevented by the combined effects of low intrinsic ubiquitin affinity, an insertion segment acting as a physical barrier across the substrate access channel, and a conformationally unstable catalytic loop in Rpn11. The docking of the structure into the proteasome EM density revealed contacts of Rpn11 with ATPase subunits, which likely stabilize the active conformation and boost the affinity for the proximal ubiquitin moiety. The narrow space around the Rpn11 active site at the entrance to the ATPase ring pore is likely to prevent erroneous deubiquitylation of folded proteins.In eukaryotes, the ubiquitin (Ub) proteasome system (UPS) is responsible for the regulated degradation of proteins (1–5). The UPS plays a key role in the maintenance of protein homeostasis by removing misfolded or damaged proteins, which could impair cellular functions, and by removing proteins whose functions are no longer needed. Consequently, the UPS is critically involved in numerous cellular processes, including cell cycle progression, apoptosis, and DNA damage repair, and malfunctions of the system often result in disease.The 26S proteasome executes the degradation of substrates that are marked for destruction by the covalent attachment of polyubiquitin chains. It is a molecular machine of 2.5 MDa comprising two subcomplexes, the 20S core particle (CP) and one or two 19S regulatory particles (RPs), which associate with the ends of the cylinder-shaped CP (6–8). The recognition and recruitment of polyubiquitylated substrates, their deubiquitylation, ATP-dependent unfolding, and translocation into the core particle take place in the RP. The structurally and mechanistically well-characterized CP houses the proteolytic activities and sequesters them from the environment, thereby avoiding collateral damage (9).The RPs attach to the outer α-rings of the CP, which control access to the proteolytic chamber formed by the inner β-subunit rings (10). Recently, the molecular architecture of the 26S holocomplex was established using cryo-EM–based approaches (11, 12), and a pseudoatomic model of the holocomplex was put forward (13). The RP is formed by two subcomplexes, known as the base and the lid, which assemble independently (12, 14). The base contains the hetero-hexameric AAA-ATPase ring (Rpt1–Rpt6), which drives the conformational changes required for substrate processing, including unfolding and translocation into the CP (15, 16). The base also contains the largest RP non-ATPase subunits, Rpn1 and Rpn2, and the Ub receptor Rpn13. The second resident Ub receptor, Rpn10, is not part of either the base or the lid; it binds only to the assembled 26S proteasome and is positioned close to the ATPase module.The lid scaffold is composed of the Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn11, and Rpn12 subunits (14). These subunits can be grouped according to their domain structures. Rpn3, Rpn5, Rpn6, Rpn7, Rpn9, and Rpn12 each comprise an N-terminal helix repeat segment, a proteasome-COP9/signalosome-eIF3 (PCI) module, and a long helix at the C terminus (8). The Rpn8 and Rpn11 subunits each consist of an Mpr1–Pad1–N-terminal (MPN) domain, followed by long C-terminal helices (Fig. 1A). The PCI subunits form a horseshoe-shaped structure and the MPN domains form a heterodimer, which are connected by a large helical bundle, to which all subunits contribute (13, 17, 18). Each of these eight subunits has paralogs in the COP9/signalosome (CSN) and the elongation initiation factor 3 (eIF3), which likely adopt a similar architecture (18–21).Open in a separate windowFig. 1.Biochemical activity of the Rpn8-Rpn11 fusion protein. (A) Domain structures of Rpn8, Rpn11 and the fusion protein. (B) Ub₄ cleavage activity of 26S proteasome, WT Rpn8-Rpn11 and Rpn8-Rpn11 (E48Q). Cleavage of labeled peptide from Ub₄ was detected by the change in fluorescence polarization after 1hr incubation at 37 °C at the indicated concentrations. Values are normalized to maximum cleavage activity of 26S proteasome. The used 26S proteasome preparation contained only trace amounts of the DUB Ubp6.The lid strengthens the interaction between the CP and RP (17) and deubiquitylates substrates before their processing by the AAA-ATPase module and the CP. Cleavage of polyubiquitin chains from the substrate enables recycling of Ub into the cellular pool, and the removal of the unfolding-resistant Ub moieties promotes translocation of substrates. The MPN domain of Rpn11 contains the catalytic site for deubiquitylation (22, 23). Rpn11 belongs to the JAB1/MPN/Mov34 metalloenzyme (JAMM) family of metalloproteases, which provide the isopeptidase activities in the proteasome, CSN, and exo-deubiquitylating enzymes (DUBs), such as associated molecule with the SH3 domain of STAM-like protein (AMSH-LP). The signature motif for this family is a conserved glutamate upstream of a zinc-coordinating catalytic loop, H(S/T)HX₇SXXD, first revealed in the structure of an archaeal homolog, AfJAMM (24). The substrate-binding mode of JAMM DUBs was clarified by the crystal structure of AMSH-LP in complex with Lys63-linked diubiquitin (25). The other proteasomal MPN subunit, Rpn8, is catalytically inactive; it does not contain the JAMM motif and appears to have mainly a supporting role for Rpn11. Isolated Rpn11 is catalytically inactive, as is the isolated lid (22). Rpn11 is activated upon integration into the 26S holocomplex and is dependent on ATP hydrolysis (23). The 26S proteasome was recently shown to undergo large-scale conformational changes from a substrate-accepting conformation to a substrate-engaged conformation that may be critical for Rpn11 function (15, 26), but the mechanistic basis for the regulation of Rpn11 remains unclear. Loss-of-function mutants of the JAMM motif cause stalling of substrates above the mouth of the ATPase module and lead to clogging of the 26S proteasome (23, 26).Inhibitors of human Rpn11 (hRpn11, also known as POH1) have been proposed as potential antitumor agents working upstream of the β5 proteolytic subunits in the UPS. The β5 subunits have been clinically validated by the approval of bortezomib and carilfzomib for the treatment of hematologic malignancies. siRNA and mutagenesis studies show that expression of the zinc catalytic domain of hRpn11 is essential for cell survival (27). Inhibition of hRpn11 in combination with EGFR inhibition has been suggested to be beneficial in the treatment of nonsmall cell lung cancer (28). Overexpression of hRpn11 in cancer cells has been linked to their tumor escape from cytotoxic agents (29). Thus, hRpn11 is an attractive target for pharmacologic intervention of the UPS.Here we present three crystal structures of the catalytically active Rpn8/Rpn11 MPN heterodimer from Saccharomyces cerevisiae, revealing the details of the Rpn11 active site and the mode of interaction with other subunits. Not all structures show proper active site geometry, hinting at possible mechanisms preventing activation outside of the proteasome complex. The access path for the C-terminal peptide of the substrate-bound Ub is blocked by a highly conserved insertion specific to Rpn11. Fitting of the Rpn8-Rpn11 crystal structure into the cryo-EM density of both the substrate-accepting and substrate-engaged proteasome revealed how the subcomplex is situated between base and PCI domain subunits, which involves long insertions unique to Rpn11 and Rpn8. Contacts to the coiled coils and the oligosaccharide-binding fold (OB) domain ring of the AAA subunits appear to control active site geometry and proper access of the isopeptide bond segment. In the substrate-engaged proteasome, the catalytic center becomes situated just above the maw of the ATPase ring.     

Association between ADHD drug use and injuries among children and adolescents

To study the association between attention deficit hyperactivity disorder (ADHD) drug use and the incidence of hospitalization due to injuries. A random sample of 150,000 persons (0–18 years) was obtained from the Dutch PHARMO record linkage system. An ADHD medication cohort as well as an up to six age/sex/index date sampled control cohort with no history of ADHD drug use was formed. Differences in incidence of hospitalization due to injuries were stratified for age and sex and compared prior, during and after exposure on ADHD drugs. The overall incidence of hospital admissions for injuries was two times higher in the ADHD medication cohort [incidence rate ratios (IRR) 2.2 (95 % CI 1.6–2.9)]. The incidence rate for injuries during exposure to ADHD drugs was lower in the exposed period compared to the period prior to ADHD drug use, although the difference was not statistically significant [IRR 0.68 (95 % CI 0.29–1.60)]. The relative risk for injuries was almost five times higher in the ADHD medication cohort among those who concomitantly used other psychotropics [IRR 4.8 (95 % CI 1.4–16.9)]. Risk for injuries was highest in 12–18 years olds. Children and adolescents using ADHD medication showed a twofold risk for hospital admissions for injuries. ADHD drug use might diminish the increased injury risk, but still overall risk is higher than in age/sex sampled children and adolescents without treatment with ADHD drugs. Use of ADHD and concomitant psychotropics increases the risk for injuries compared to only ADHD drug use.     

Heterogeneity of proteoglycans extracted before and after collagenase treatment of human articular cartilage: I. Physical properties related to age

Proteoglycans were isolated from young and mature human articular cartilage 4 different ways: by direct extraction with 4M guanidine hydrochloride (GuHCl); after digestion of the residue from this first extraction with collagenase, by extraction with 4M GuHCl; associatively with 0.5M GuHCl after digestion of the cartilage with collagenase; and dissociatively with 4M GuHCl after digestion of the cartilage with collagenase. The structural properties of these proteoglycans were compared. Proteoglycan aggregates and monomers isolated from second extractions and from young cartilage were of larger hydrodynamic size than proteoglycans isolated from first extractions and mature cartilage, respectively. The same applied to the chondroitin sulfate chain lengths of these proteoglycans. The proteoglycan fraction from second extractions of cartilage contained a larger proportion of monomers than the fraction from first extractions. Associative extraction of mature collagenase-digested cartilage yielded mainly proteoglycan monomers, whereas an appreciable amount of proteoglycan aggregate was also liberated from young collagenase-digested cartilage. Our results indicate that, because of their larger size, proteoglycans from second extractions of cartilage are more entrapped in the collagen network. These large proteoglycans can only be liberated from the matrix after extraction of the smaller proteoglycans, followed by digestion of the residue with collagenase. This indicates that proteoglycans overlap and entangle with the collagen and protect it from degradation by collagenase.     

Normal viability and altered pharmacokinetics in mice lacking mdr1-type (drug-transporting) P-glycoproteins

The mdr1-type P-glycoproteins (P-gps) confer multidrug resistance to cancer cells by active extrusion of a wide range of drugs from the cell. To study their physiological roles, we have generated mice genetically deficient in the mdr1b gene [mdr1b (−/−) mice] and in both the mdr1a and mdr1b genes [mdr1a/1b (−/−) mice]. In spite of the host of functions speculatively attributed to the mdr1-type P-gps, we found no physiological abnormalities in either strain. Viability, fertility, and a range of histological, hematological, serum–chemical, and immunological parameters were not abnormal in mdr1a/1b (−/−) mice. The high level of mdr1b P-gp normally present in the pregnant uterus did not protect fetuses from a drug (digoxin) in the bloodstream of the mother, although the protein did reduce drug accumulation in the adrenal gland and ovaries. Pharmacologically, mdr1a/1b (−/−) mice behaved similarly to the previously analyzed mdr1a (−/−) mice, displaying, for instance, increased brain penetration and reduced elimination of digoxin. However, both mdr1a and mdr1b P-gps contributed to the extrusion of rhodamine from hematopoietic progenitor cells, suggesting a potential role for the endogenous mdr1-type P-gps in protection of bone marrow against cytotoxic anticancer drugs. This, and the normal viability of mdr1a/1b (−/−) mice, has implications for the use of P-gp-blocking agents in cancer and other chemotherapy. mdr1a/1b (−/−) mice should provide a useful model system to further test the pharmacological roles of the drug-transporting P-gps and to analyze the specificity and effectivity of P-gp-blocking drugs.