Karen Meerovitch

Identification and Characterization of 1,25-Dihydroxyvitamin D3-responsive Repressor Sequences in the Rat Parathyroid Hormone-related Peptide Gene

Parathyroid hormone-related peptide (PTHRP) gene transcription is suppressed by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3. In the present report, we examined 1,25(OH)2D3-mediated repression of PTHRP expression by transfection of PTHRP promoter/reporter constructs in normal human keratinocytes and by DNA binding. We localized an element conferring 1,25(OH)2D3-mediated repression in vivo to a 47-base pair (bp) region located −1121 to −1075 from the transcriptional start site. Mobility shift analysis revealed that this vitamin D response element (VDRE) forms DNA-protein complexes. The addition of a monoclonal antibody that recognizes the DNA binding region of the vitamin D receptor (VDR) attenuated binding of the receptor to the 47-bp sequence, whereas the addition of monoclonal antibody raised against the retinoid X receptor (RXR) further retarded the mobility of the protein-DNA complex. Consequently, the PTHRP promoter element binds a VDR·RXR heterodimer. Examination of this VDRE revealed complete sequence homology with a half-site of the human and rat osteocalcin VDRE (GGGTGA). Furthermore, mutation analysis suggests that a 16-bp domain consisting of an almost perfect repeat separated by a 3-base pair “spacer” GAG is responsible for the DNA-protein interaction within this 47-bp sequence. Our results therefore indicate the existence of an inhibitory VDRE within the PTHRP promoter that is similar in sequence composition and cellular factor requirement to classical up-regulatory VDREs.

Hepatitis C virus NS2/3 processing is required for NS3 stability and viral RNA replication

The hepatitis C virus NS2/3 protease is responsible for cleavage of the viral polyprotein between nonstructural proteins NS2 and NS3. We show here that mutation of three highly conserved residues in NS2 (His952, Glu972, and Cys993) abrogates NS2/3 protease activity and that introduction of any of these mutations into subgenomic NS2-5B replicons results in complete inactivation of NS2/3 processing and RNA replication in both stable and transient replication assays. The effect of uncleaved NS2 on the various activities of NS3 was therefore explored. Unprocessed NS2 had no significant effect on the in vitro ATPase and helicase activities of NS3, whereas immunoprecipitation experiments demonstrated a decreased affinity of NS4A for uncleaved NS2/3 as compared with NS3. This subsequently resulted in reduced kinetics in an in vitro NS3 protease assay with the unprocessed NS2/3 protein. Interestingly, NS3 was still capable of efficient processing of the polyprotein expressed from a subgenomic replicon in Huh-7 cells in the presence of uncleaved NS2. Notably, we show that fusion with NS2 leads to the rapid degradation of NS3, whose activity is essential for RNA replication. Finally, we demonstrate that uncleaved NS2/3 degradation can be prevented by the addition of a proteasome inhibitor. We therefore propose that NS2/3 processing is a critical step in the viral life cycle and is required to permit the accumulation of sufficient NS3 for RNA replication to occur. The regulation of NS2/3 cleavage could constitute a novel mechanism of switching between viral RNA replication and other processes of the hepatitis C virus life cycle.

Proparathyroid Hormone-related Protein Is Associated with the Chaperone Protein BiP and Undergoes Proteasome-mediated Degradation

Parathyroid hormone-related peptide (PTHrP) is an important causal factor for hypercalcemia associated with malignancy. In addition to the endocrine functions attributed to secretory forms of the peptide, PTHrP also plays a local role as a mediator of cellular growth and differentiation presumably at least in part through intracellular pathways. In studying the post-translational regulation of PTHrP, we observed that PTHrP was conjugated to multiple ubiquitin moieties. We report here that the proteasome is responsible for the degradation of the endoplasmic reticulum-associated precursor, pro-PTHrP. Cells expressing prepro-PTHrP and exposed to lactacystin accumulate pro-PTHrP assessed by anti-pro specific antibodies. Brefeldin A-treated cells also accumulate pro-PTHrP suggesting that degradation does not occur in the endoplasmic reticulum (ER) lumen. Subcellular fractionation of both lactacystin and brefeldin A-treated cells indicated that accumulated pro-PTHrP resides in microsomal fractions with a portion of the protein exposed to the cytosolic side of the ER membrane as assessed by protease protection experiments. Immunoprecipitation and Western blot analysis identified pro-PTHrP in association with the ER molecular chaperone protein BiP. We conclude that pro-PTHrP from the ER can gain access to the cytoplasmic side of the ER membrane where it can undergo ubiquitination and degradation by the proteasome.

Safety and efficacy of MIM-D3 ophthalmic solutions in a randomized, placebo-controlled Phase 2 clinical trial in patients with dry eye.

Purpose To evaluate the safety and efficacy of ophthalmic MIM-D3, a tyrosine kinase TrkA receptor agonist, in patients with dry eye. Design A prospective, two-center, randomized, double-masked, placebo-controlled Phase 2 study. Methods A total of 150 dry eye patients were randomized 1:1:1 to study medication (1% MIM-D3, 5% MIM-D3, or placebo) and dosed twice daily (BID) for 28 days. Key eligibility criteria included exacerbation in corneal staining and ocular discomfort in the Controlled Adverse Environment (CAESM) on two visits, separated by 1 week of BID dosing with artificial tears. Safety and efficacy were evaluated at baseline, throughout treatment, and for 2 weeks post-treatment. The pre-specified primary outcome measures were fluorescein corneal staining post-CAE at day 28 and diary worst symptom scores over 28 days. Secondary outcomes included the pre-, post-, and the change from pre- to post-CAE fluorescein and lissamine green staining in both corneal and conjunctival regions, as well as individual diary symptoms. Results The prespecified primary endpoints were not met. Compared with placebo, fluorescein corneal staining at day 28 was significantly improved (P < 0.05) in the 1% MIM-D3 group for the assessment of change from pre-CAE to post-CAE. In addition, following CAE exposure, patients in the 1% MIM-D3 group showed significant improvements versus placebo (P < 0.05) in inferior fluorescein and lissamine green staining after 14 and 28 days. Compared with placebo, patients in the 5% MIM-D3 group reported significantly lower daily diary scores for ocular dryness (P < 0.05). In a subgroup defined by higher symptom scores during the run-in period, significant treatment effects (P < 0.05) were observed for diary symptoms for both MIM-D3 doses. Ocular adverse events were mild and not considered to be treatment-related. Conclusion Treatment with topical ophthalmic MIM-D3 demonstrated protection against the effects of a CAE challenge on dry eye signs, reduced patient-reported diary symptoms, with a favorable safety profile.

Preproparathyroid Hormone-related Protein, a Secreted Peptide, Is a Substrate for the Ubiquitin Proteolytic System

Parathyroid hormone-related protein (PTHrP) is an important causal factor of hypercalcemia associated with malignancy. PTHrP also modulates cell growth and differentiation of normal cells through mechanisms that include binding to cell surface-specific receptors as well as by possible intracellular routes. To understand the regulation of intracellular PTHrP expression, post-translational processing of PTHrP was investigated. Using cell-free translations it was shown that PTHrP can be ligated efficiently to multiple ubiquitin moieties. Both conjugation to ubiquitin and degradation of prepro-PTHrP synthesized in vitro were ATP-dependent. Translation in vitro in the presence of the proteasome inhibitor MG-132 abolished the degradation of PTHrP. Treatment of cells, cotransfected with hemagglutinin-tagged ubiquitin and histidine-tagged prepro-PTHrP, with MG-132, led to the accumulation of ubiquitinated prepro-PTHrP. Deletion mutagenesis experiments indicated that both the prepro secretory domain and a PEST (amino acid residues Pro (P), Glu (E), and/or Asp (D), Ser (S), and Thr (T)) motif in the COOH-terminal region of the protein were not required as cis-acting determinants for ubiquitination. This is the first report of a wild-type secretory polypeptide serving as a substrate of the ubiquitin proteolytic pathway. These results suggest that the ubiquitin-dependent proteolytic pathway is involved in regulating the metabolic stability of intracellular PTHrP, and this regulation may be an important mechanism for modulating its effects on cell growth and differentiation.

Small-Molecule Ligands of GD2 Ganglioside, Designed from NMR Studies, Exhibit Induced-Fit Binding and Bioactivity

Ganglioside GD2 is a cell surface glycosphingolipid. Targeting of GD2, i.e., by anti-GD2 mAb 3F8, is used clinically for cancer diagnosis, prognosis, and therapy. Here, the conformations of free GD2, and of GD2 bound to mAb 3F8, were resolved by saturation transfer difference NMR and molecular modeling. Then, three small-molecule cyclic peptide ligands that bind to GD2 selectively were designed. Transferred nuclear Overhauser enhancement of the GD2-bound conformation of the peptide ligands showed an induced-fit binding mechanism. The mAb 3F8 and the peptidic GD2 ligands mediate similar biological functions in cell-based assays of calcium fluxes and src activation. Thus, small molecules can selectively and functionally interact with a sugar head group. This work furthers the concept of rationally designing ligands for carbohydrate targets, and may be expanded to other clinically relevant gangliosides.

Studies on the Mechanism of Internal Initiation of Translation on Poliovirus RNA

The genome of the picornaviruses, of which poliovirus is a member, is a plus-sense single stranded RNA molecule. Unlike cellular mRNAs, the 5′ end of picornavirus mRNAs is not capped, but instead is terminated with pUp (Hewlett et al., 1976; Nomoto et al., 1976). The 5′ untranslated region (UTR) is unusually long (approximately 750 nucleotides for poliovirus) and highly structured (for recent review see Agol, 1991). In addition, the 5′ UTR contains multiple upstream AUGs (6–8 for poliovirus), none of which appear to be used as initiators of translation (Pelletier et al., 1988a). Because poliovirus RNA is naturally uncapped and translates in vivo and in vitro under conditions where cap-dependent translation is impaired, it must translate by a cap-independent mechanism. Consistent with this, an internal sequence in the poliovirus mRNA 5′ UTR (spanning approximately 450 nucleotides) is sufficient for cap-independent translation (Pelletier et. al., 1988b), and this sequence can also confer cap-independent translation to heterologous mRNAs (Pelletier et. al., 1988b; Trono et al., 1988). This region serves as an internal binding site for ribosomes and is termed Ribosome Landing Pad (RLP) (Pelletier and Sonenberg, 1988c). Internal initiation of translation also occurs for other members of the picornavirus group (e.g. Jang et al., 1988, for review see Jackson et al., 1990), plant viral mRNAs (Thomas et al., 1991) and for several cellular mRNAs (Macejak and Sarnow, 1991; Oh et al., 1992).

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.