Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis

Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp³⁴² and Asn³⁴³. Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.     

A Single Conserved Residue Mediates Binding of the Ribonucleotide Reductase Catalytic Subunit RRM1 to RRM2 and Is Essential for Mouse Development

The ribonucleotide reductase (RNR) complex, composed of a catalytic subunit (RRM1) and a regulatory subunit (RRM2), is thought to be a rate-limiting enzymatic complex for the production of nucleotides. In humans, the Rrm1 gene lies at 11p15.5, a tumor suppressor region, and RRM1 expression in cancer has been shown to predict responses to chemotherapy. Nevertheless, whether RRM1 is essential in mammalian cells and what the effects of its haploinsufficiency are remain unknown. To model RNR function in mice we used a mutation previously described in Saccharomyces cerevisiae (Rnr1-W688G) which, despite being viable, leads to increased interaction of the RNR complex with its allosteric inhibitor Sml1. In contrast to yeast, homozygous mutant mice carrying the Rrm1 mutation (Rrm1^WG/WG) are not viable, even at the earliest embryonic stages. Proteomic analyses failed to identify proteins that specifically bind to the mutant RRM1 but revealed that, in mammals, the mutation prevents RRM1 binding to RRM2. Despite the impact of the mutation, Rrm1^WG/+ mice and cells presented no obvious phenotype, suggesting that the RRM1 protein exists in excess. Our work reveals that binding of RRM1 to RRM2 is essential for mammalian cells and provides the first loss-of-function model of the RNR complex for genetic studies.     

Important Role for the Murid Herpesvirus 4 Ribonucleotide Reductase Large Subunit in Host Colonization via the Respiratory Tract

Viral enzymes that process small molecules provide potential chemotherapeutic targets. A key constraint—the replicative potential of spontaneous enzyme mutants—has been hard to define with human gammaherpesviruses because of their narrow species tropisms. Here, we disrupted the murid herpesvirus 4 (MuHV-4) ORF61, which encodes its ribonucleotide reductase (RNR) large subunit. Mutant viruses showed delayed in vitro lytic replication, failed to establish infection via the upper respiratory tract, and replicated to only a very limited extent in the lower respiratory tract without reaching lymphoid tissue. RNR could therefore provide a good target for gammaherpesvirus chemotherapy.Cellular deoxyribonucleotide synthesis is strongly cell cycle dependent. DNA viruses replicating in noncycling cells must therefore either induce cellular enzymes or supply their own. Most herpesviruses encode multiple homologs of nucleotide metabolism enzymes, including both subunits of the cellular ribonucleotide reductase (RNR) (4). While most in vivo cells are resting, most in vitro cell lines divide continuously (29). The importance of viral RNRs may therefore only be apparent in vivo (14). In contrast to alpha- and betaherpesviruses, gammaherpesviruses cause disease mainly through latency-associated cell proliferation. However, gamma-2 herpesviruses show lytic gene expression in sites of latency (9, 17), and lytic reactivation could potentially alleviate some gammaherpesvirus-infected cancers (7, 8). Therefore, it is important also to understand the pathogenetic roles of gammaherpesvirus lytic cycle enzymes, such as RNR.The known human gammaherpesviruses Epstein-Barr virus (EBV) and Kaposi''s sarcoma-associated herpesvirus (KSHV) have narrow species tropisms that preclude most pathogenesis studies. In contrast, murid herpesvirus 4 (MuHV-4) (21, 26) allows gammaherpesvirus host colonization to be studied in vivo. After intranasal (i.n.) inoculation, MuHV-4 replicates lytically in lung epithelial cells before seeding to lymphoid tissue (27). Long-term virus loads are independent of extensive primary lytic spread (25). However, whether persistence requires some lytic gene expression remains unclear. Replication-deficient viral DNA reached the spleen after intraperitoneal (i.p.) but not i.n. virus inoculation (15, 20, 28), suggesting that virus dissemination from the lung to lymphoid tissue requires lytic replication. In addition, less invasive inoculations may increase further the viral functions required to establish a persistent infection. Thymidine kinase (TK)-deficient MuHV-4 given i.n. without general anesthesia, in which method the wild-type virus infects the upper respiratory tract and reaches lymphoid tissue without infecting the lungs (18), fails to colonize in mice at all (12). The implication is that virions using a likely physiological route of host entry must replicate in terminally differentiated cells to establish a significant infection. However, some unusual features of gammaherpesvirus TKs (11) suggest that they have functions besides thymidine phosphorylation. We therefore targeted here another enzyme linked to viral DNA replication, the MuHV-4 RNR. We aimed to define the in vivo importance of a potential therapeutic target and to advance generally our understanding of gammaherpesvirus pathogenesis.Transposon insertions in the MuHV-4 RNR small (ORF60) and large (ORF61) RNR subunit genes have been described as either attenuating or not for lytic replication in vitro (19, 23). We disrupted ORF61 (RNR⁻) by inserting stop codons close to its 5′ end (Fig. ​(Fig.11 a). An EcoRI-L genomic clone (coordinates 80644 to 84996) in pUC19 (6) was digested with AleI to remove nucleotides 82320 to 82534 of ORF61 (82865 to 80514). An oligonucleotide encoding multiple stop codons and an EcoRI restriction site (5′-CTAGCATGCTAGAATTCTAGCATGCATG-3′) was ligated in place. Nucleotides 81365 to 83883 were then PCR amplified, including a BamHI site in the 81365 primer, cloned as a BglII/BamHI fragment into the BamHI site of pST76K-SR, and recombined into a MuHV-4 bacterial artificial chromosome (BAC) (1). A revertant virus was made by reconstituting the corresponding, unmutated genomic fragment. Southern blots (5) of viral DNA (Fig. ​(Fig.1b)1b) confirmed the expected genomic structures, and immunoblots (5) of infected cell lysates (Fig. ​(Fig.1c)1c) established that mutant viruses no longer expressed the RNR large subunit.Open in a separate windowFIG. 1.Disruption of the MuHV-4 ORF61. (a) Schematic diagram of the ORF61 (RNR large) locus, showing the mutation introduced and relevant restriction sites. (b) Viral DNA was digested with EcoRI and probed for ORF61. Oligonucleotide insertion into ORF61 changes a 4,352-bp wild-type band to 2,462 bp plus 1,676 bp. The 2,462-bp fragment is not visible because it overlaps the probe by only 331 nucleotides (nt) and comigrates with a background band of unknown origin. WT, wild type; REV, revertant; RNR⁻, mutant; RNR⁻ ind, independent mutant. WT luc⁺ is MuHV-4 expressing luciferase from an ORF57/ORF58 intergenic cassette. RNR⁻ luc⁺ and RNR⁻ luc⁺ind have ORF61 disrupted on this background. (c) Infected cell lysates were immunoblotted for gp150 (virion envelope glycoprotein, monoclonal antibody [MAb] T1A1), ORF17 (capsid component, MAb 150-7D1), TK (tegument component, MAb CS-4A5), and ORF61 (MAb PS-8A7). (d) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (0.01 eGFP units/cell, 2 h, 37°C), washed two times with phosphate-buffered saline (PBS) to remove unbound virions, and cultured at 37°C to allow virus spread. Infectivity (in eGFP units) at each time point was determined on fresh BHK-21 cells in the presence of phosphonoacetic acid to prevent further viral spread, with the number of eGFP-postive cells counted 18 h later by flow cytometry. (e) BHK-21 cells were infected with RNR⁺ or RNR⁻ viruses (2 eGFP units/cell, 2 h, 37°C), washed in medium (pH 3) to inactivate nonendocytosed virions, and cultured at 37°C to allow virus replication. The infectivity of replicate cultures was then assayed as described in the legend of panel d. (f) BHK-21 cells were incubated with RNR⁺ or RNR⁻ viruses (0.3 eGFP units/cell, 37°C) for the times indicated, and the numbers of eGFP-positive cells in the cultures were then determined by flow cytometry.RNR⁻ viruses were noticeably slower than RNR⁺ viruses when spreading through BHK-21 cell monolayers after BAC DNA transfection. Normalizing by immunoblot signal, RNR⁻ virus stocks had titers similar to that of the wild type by viral enhanced green fluorescent protein (eGFP) expression but 10- to 100-fold lower plaque titers. Using eGFP expression as a readout, RNR⁻ virion production after a low multiplicity of infection lagged 1 day behind that of the wild type (Fig. ​(Fig.1d).1d). Maximum infectivity yields were also reduced, but once BHK-21 cells become confluent, they support MuHV-4 lytic infection poorly, so this was probably a consequence of the slower lytic spread. After a high multiplicity of infection (Fig. ​(Fig.1e),1e), RNR⁻ mutants showed a 10-h lag in virion production and no difference in the final yield. They showed no defect in single-cycle eGFP expression (Fig. ​(Fig.1f),1f), implying normal virion entry. Therefore, the main RNR⁻ defect lay in infectious virion production.For in vivo experiments, the loxP-flanked viral BAC-eGFP cassette must be removed (1). Therefore, to monitor infection in vivo without having to rely on new virion production as a readout, we transferred the RNR mutation onto a luciferase-positive (luc⁺) background (18). Viral luciferase expression (from an early lytic promoter) by in vitro luminometry (18) was independent of either viral DNA replication or RNR expression (Fig. ​(Fig.22 a). After i.n. inoculation of anesthetized mice, RNR⁻ luciferase signals measured in vivo by i.p. luciferin injection and IVIS Lumina charge-coupled-device (CCD) camera scanning (18) were visible in lungs (Fig. ​(Fig.2b)2b) but were 100-fold lower than those of the RNR⁺ controls (Fig. ​(Fig.2c).2c). A severe impairment of RNR⁻ lytic replication was confirmed by plaque assay (18) (Fig. ​(Fig.2d);2d); the difference between RNR⁻ and RNR⁺ plaque titers greatly exceeded any difference in plaquing efficiency.Open in a separate windowFIG. 2.Host colonization by RNR⁻ MuHV-4 mutants. (a) BHK-21 cells were left uninfected or infected overnight with RNR⁺ or RNR⁻ luc⁺ MuHV-4 and then assayed for luciferase expression by luminometry. Phosphonoacetic acid (PAA; 100 μg/ml) was either added or not to cultures to block viral late gene expression. Each point shows the mean ± standard deviation from triplicate cultures. (b) BALB/c mice were infected i.n. under general anesthesia with RNR⁻ or RNR⁺ luc⁺ MuHV-4 (5 × 10³ PFU) and then assayed for luciferase expression by luciferin injection and CCD camera scanning. The images are from 5 days postinfection. Note that the RNR⁺ and RNR⁻ images have different sensitivity scales. (c) For quantitation, dorsal and ventral luciferase signals were summed. Each point shows 1 mouse. The dashed lines show detection thresholds. The RNR⁺ signal was significantly greater than the RNR⁻ signal for all sites and time points (P < 0.001 by Student''s t test). (d) C57BL/6 mice were infected i.n. under anesthesia with RNR⁻ or RNR⁺ MuHV-4 (5 × 10³ PFU). Five days later, infectious virus loads in noses and lungs were measured by plaque assay. Each point shows 1 mouse. RNR⁻ infections yielded no plaques and therefore are shown at the sensitivity limits of each assay. (e) BALB/c mice were infected i.n. with RNR⁻ or RNR⁺ MuHV-4 without anesthesia and then monitored by luciferin injection and CCD camera scanning. Each point shows the summed ventral and dorsal signals of the relevant region for 1 mouse. Neck signals correspond to the superficial cervical lymph nodes (SCLN). The dashed lines show detection thresholds. RNR⁻ luciferase signals were undetectable at all time points.No RNR⁻ luciferase signals were visible in noses, nor did RNR⁻ MuHV-4 give signals in the superficial cervical lymph nodes (SCLN), which drain the nose (Fig. ​(Fig.2c).2c). This lack of live imaging signals from the upper respiratory tract was confirmed by ex vivo imaging of SCLN at day 14 postinfection. We examined upper respiratory tract infection further with an independently derived luc⁺ RNR⁻ mutant, inoculating i.n. without anesthesia so as to avoid virus aspiration into the lungs. No RNR⁻ luciferase signals were detected, while wild-type signals were readily observed in the nose and superficial cervical lymph nodes (Fig. ​(Fig.2e2e).Like RNR⁻ MuHV-4, TK⁻ mutants are severely attenuated for lytic replication in the lower respiratory tract. However, they eventually establish a reactivatable latent infection and induce virus-specific antibody (3). Latent virus titers in spleens peak at 1 month postinoculation. Infectious center assays showed no RNR⁻ infection of spleens at that time (Fig. ​(Fig.33 a). We also looked for viral DNA in spleens by quantitative PCR (Fig. ​(Fig.3b).3b). Genomic coordinates 4166 to 4252 were amplified and hybridized to a probe with coordinates 4218 to 4189. Viral genome copies, relative to the cellular adenosine phosphoribosyl transferase copy number, were calculated from standard curves of cloned plasmid DNA (10). No RNR⁻ viral DNA was detected. ELISA for MuHV-4-specific serum IgG (24) detected an antibody response after lung infection but not upper respiratory tract infection of BALB/c mice with RNR⁻ MuHV-4 (Fig. ​(Fig.3c).3c). There was a similar lack of antibody 1 month after upper respiratory tract infection of C57BL/6 mice with independently derived RNR⁻ mutants (Fig. ​(Fig.3d)3d) and 3 months after exposure of 6 BALB/c mice to RNR⁻ luc⁺ MuHV-4. In contrast, i.p. RNR⁻ luc⁺ MuHV-4 gave lower luciferase signals than RNR⁺ luc⁺ MuHV-4 (Fig. ​(Fig.44 a), but RNR⁻ infectious centers (Fig. ​(Fig.4b)4b) and viral genomes (Fig. ​(Fig.4c)4c) were detected in spleens, and enzyme-linked immunosorbent assays (ELISAs) (Fig. ​(Fig.4d)4d) showed MuHV-4-specific serum IgG.Open in a separate windowFIG. 3.Spleen colonization by RNR⁻ MuHV-4. (a) BALB/c or C57BL/6 mice were infected i.n. either with general anesthesia (lung infection) or without (nose infection). One month later, spleens were assayed for recoverable latent virus by infectious center assay. Lower detection limit, 10 infectious centers per spleen. (b) The spleens described in the legend of panel a were further analyzed for viral DNA by quantitative PCR. Copy numbers are expressed relative to the cellular adenosine phosphoribosyl transferase copy number in each sample. The dashed lines show lower detection limits (1 viral copy/10,000 cellular copies). (c) Sera from BALB/c mice after i.n. infection either with (lung infection) or without (nose infection) general anesthesia were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance curve for 1 mouse. The dashed lines show naive serum. (d) Sera from C57BL/6 mice 1 month after infection with independent RNR mutants were analyzed for MuHV-4-specific IgG, as described in the legend to panel c.Open in a separate windowFIG. 4.Intraperitoneal infection with RNR⁺ and RNR⁻ MuHV-4. (a) Mice were infected i.p. with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 and then monitored for luciferase expression. Each point shows the total abdominal signal of 1 mouse. The x axis is at the lower limit of signal detection above the background. (b) Spleens were assayed for recoverable virus by infectious center assay 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4. Each point shows the titer of 1 mouse. One log₁₀ infectious center per mouse corresponds to the lower limit of detection. (c) Spleen DNA was analyzed for viral genome content by quantitative PCR. Each point shows viral copy/cellular copy for the mean of triplicate reactions for 1 mouse. (d) Sera taken 10 days after i.p. infection with RNR⁻ luc⁺ or RNR⁺ luc⁺ MuHV-4 were assayed for MuHV-4-specific IgG by ELISA. Each line shows the absorbance values for the serum of 1 mouse. “Naive” represents age-matched, uninfected controls.The failure of both the RNR large subunit (ORF61) and TK⁻ MuHV-4 mutants to infect via the upper respiratory tract argues that this requires viral replication in a nucleotide-poor cell. The additional lack of lymphoid RNR⁻ infection after inoculation into the lungs seemed likely to reflect a defect in virus transport, as RNR⁻ MuHV-4 did colonize the spleen after i.p. inoculation. It is also possible that the first cells infected simply produced no infectious virions, although this seemed a more likely explanation for upper respiratory tract infection being undetectable; lung infection progressed sufficiently to give detectable luciferase expression and to induce an antiviral antibody response. How transport from lung to lymphoid tissue occurs is unknown, but likely scenarios include latently infected dendritic cells (22) carrying MuHV-4 along afferent lymphatics to germinal centers and cell-free virions being captured in lymph nodes by subcapsular sinus macrophages (13). Therefore, RNR may be important for MuHV-4 to spread from myeloid cells to B cells.The difference between RNR⁻ and TK⁻ mutants in host colonization via the lung—TK⁻ mutants reached lymphoid tissue whereas RNR⁻ mutants did not—could reflect additional ORF61 functions, as precedent exists for functional drift (2, 16). Alternatively, RNR may be needed more than TK for MuHV-4 replication in some cell types. Formidable hurdles to RNR-based therapies remain: human gammaherpesvirus infections rarely present until latency is well established, so blocking virus spread to lymphoid tissue may have a limited impact, and no drugs sufficiently selective to target viral RNRs in a clinical setting have yet emerged. Nevertheless, the severe in vivo attenuation of RNR⁻ MuHV-4 suggested that RNR may be a viable target for limiting gammaherpesvirus lytic spread.     

Rice stripe1-2 and stripe1-3 Mutants Encoding the Small Subunit of Ribonucleotide Reductase Are Temperature Sensitive and Are Required for Chlorophyll Biosynthesis

We induced mutants, stripe1-2 (st1-2) and stripe1-3 (st1-3), from rice (Oryza sativa L.) Indica 9311 using Ethyl methanesulfonate (EMS). Both st1-2 and st1-3 mutants encoded the small subunit of ribonucleotide reductase 1 (RNRS1), differed in the location of the mutated base, and displayed white-stripe from the L2 stage through maturity. The mutants were sensitive to temperature, and their chlorophyll content increased with the increase in temperature; however, they did not revert to normal green leaf phenotype under field conditions. The mutant st1-2 showed loosely arranged thylakoid lamellar structure as compared with wild-type (WT) plants. Contrastingly, st1-3 displayed normal thylakoid lamellar structure, good agronomic traits, and higher yield than st1-2 but lower yield than WT. Three-dimensional structure prediction for RNRS1 indicated that the mutation in Val-171 residue in st1-2 influenced the connection of RNRS1 to iron, causing abnormal development of chloroplasts. Real-time PCR analysis showed that the expression levels associated with chlorophyll biosynthetic pathway and photosynthesis were affected in st1-2 and st1-3 at different temperatures and different developmental stages.     

Detection of a Stable Free Radical in the B2 Subunit of the Manganese Ribonucleotide Reductase (MN-RRase) of Corynebacterium ammoniagenes

Ribonucleotide reductases catalyze the irreversible reductive formation of 2′-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Coynebacterium ammoniagenes ATCC 6872. Enrichment by 2′5′-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with Superose 12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hy-droxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa 82 subunit of the Mn-RRase of Corynebacteriurn ammoniagenes.     

Ribonucleotide Reductase Large Subunit M1 Predicts Poor Survival Due to Modulation of Proliferative and Invasive Ability of Gastric Cancer

Objectives

We aimed to investigate the prognostic value of RRM1 in GC patients.

Methods

A total of assessable 389 GC patients with clinicopathological and survival information were enrolled from City of Hope (COH, n = 67) and Zhejiang University (ZJU, n = 322). RRM1 protein expression was determined by immunohistochemistry on FFPE tissue samples. Kaplan-Meier and Cox analyses were used to measure survival. Ras/Raf activity and invasion assays were used to evaluate the role of RRM1 in GC cell lines.

Results

In vitro experiments demonstrated RRM1 activated Ras/Raf/MAPK signal transduction and promoted GC cell proliferation. Meanwhile, RRM1 expression was significantly associated with lymph node involvement, tumor size, Ki67 expression, histological subtype and histological grade in the GC tissue samples (p<0.05). Kaplan-Meier analysis illustrated that high RRM1 expression predicted poor survival in GC patients in the COH and ZJU cohorts (log-rank p<0.01). In multivariate Cox analysis, the hazard ratios of RRM1 for overall survival were 2.55 (95% CI 1.27–5.15) and 1.51 (95% CI 1.07–2.13) in the COH and ZJU sets, respectively. In particular, RRM1 specifically predicted the outcome of advanced GCs with poor differentiation and high proliferative ability. Furthermore, inhibition of RRM1 by siRNA significantly reduced the dNTP pool, Ras/Raf and MMP-9 activities and the levels of p-MEK, p-ERK and NF-κB, resulting in growth retardation and reduced invasion in AGS and NCI-N87 cells.

Conclusions

RRM1 overexpression predicts poor survival in GC patients with advanced TNM stage. RRM1 could potentially serve as prognostic biomarker and therapeutic target for GCs.     

The Conserved Lys-95 Charged Residue Cluster Is Critical for the Homodimerization and Enzyme Activity of Human Ribonucleotide Reductase Small Subunit M2

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G₁/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.     

A Novel Clade of Unique Eukaryotic Ribonucleotide Reductase R2 Subunits is Exclusive to Apicomplexan Parasites

Apicomplexa are protist parasites of tremendous medical and economic importance, causing millions of deaths and billions of dollars in losses each year. Apicomplexan-related diseases may be controlled via inhibition of essential enzymes. Ribonucleotide reductase (RNR) provides the only de novo means of synthesizing deoxyribonucleotides, essential precursors for DNA replication and repair. RNR has long been the target of antibacterial and antiviral therapeutics. However, targeting this ubiquitous protein in eukaryotic pathogens may be problematic unless these proteins differ significantly from that of their respective host. The typical eukaryotic RNR enzymes belong to class Ia, and the holoenzyme consists minimally of two R1 and two R2 subunits (α₂β₂). We generated a comparative, annotated, structure-based, multiple-sequence alignment of R2 subunits, identified a clade of R2 subunits unique to Apicomplexa, and determined its phylogenetic position. Our analyses revealed that the apicomplexan-specific sequences share characteristics with both class I R2 and R2lox proteins. The putative radical-harboring residue, essential for the reduction reaction by class Ia R2-containing holoenzymes, was not conserved within this group. Phylogenetic analyses suggest that class Ia subunits are not monophyletic and consistently placed the apicomplexan-specific clade sister to the remaining class Ia eukaryote R2 subunits. Our research suggests that the novel apicomplexan R2 subunit may be a promising candidate for chemotherapeutic-induced inhibition as it differs greatly from known eukaryotic host RNRs and may be specifically targeted.     

Identification of 'Non-Proliferating' B16 Melanoma Cells Using Monoclonal Antibody (Ad203) Against the M1 Subunit of Ribonucleotide Reductase

Abstract. Ribonucleotide reductase catalyses a critical reaction in DNA synthesis. Its M1 subunit is present during all proliferative phases of the cell cycle, but apparently not in the quiescent phase Go. We have used a monoclonal antibody (AD203) directed against the M1 subunit to distinguish immunocytochemically proliferating from non-proliferating cultured B16 mouse melanoma cells during exponential growth. the presumption that AD203 unstained cells constituted a non-proliferating fraction was tested by simultaneously counting the cells that failed to incorporate bromodeoxyuridine (BrdU) into DNA during a prolonged BrdU exposure. the proportion of cells which did not incorporate BrdU was found to correlate closely with the proportion not staining with AD203 and therefore presumably lacking the M1 subunit. the respective morphological features of AD203 stained and unstained cells were found not to differ significantly.     

Ribonucleotide Reductase Subunit M2 Predicts Survival in Subgroups of Patients with Non-Small Cell Lung Carcinoma: Effects of Gender and Smoking Status

BackgroundRibonucleotide reductase catalyzes the conversion of ribonucleotide diphosphates to deoxyribonucleotide diphosphates. The functional enzyme consists of two subunits - one large (RRM1) and one small (RRM2 or RRM2b) subunit. Expression levels of each subunit have been implicated in prognostic outcomes in several different types of cancers.ResultsIn non-small cell lung cancer (NSCLC), RRM2 expression was strongly predictive of disease-specific survival in women, non-smokers and former smokers who had quit at least 10 years prior to being diagnosed with lung cancer. Higher expression was associated with worse survival. This was not the case for men, current smokers and those who had stopped smoking for shorter periods of time. RRM1 was not predictive of survival outcomes in any subset of the patient group.ConclusionRRM2, but not RRM1, is a useful predictor of survival outcome in certain subsets of NSCLC patients.     

The Plausibility of RNA-Templated Peptides: Simultaneous RNA Affinity for Adjacent Peptide Side Chains

According to the RNA world hypothesis, coded peptide synthesis (translation) must have been first catalyzed by RNAs. Here, we show that small RNA sequences can simultaneously bind the dissimilar amino acids His and Phe in peptide linkage. We used in vitro counterselection/selection to isolate a pool of RNAs that bind the dipeptide NH(2)-His-Phe-COOH with K (D) ranging from 36 to 480 μM. These sites contact both side chains, usually including the protonated imidazole of His, but bind-free L: -His and L: -Phe with much lower, sometimes undetectable, affinities. The most frequent His-Phe sites do not usually contain previously isolated sites for individual amino acids, and are only ≈35 % larger than previously known separate His and Phe sites. Nonetheless, His-Phe sites appear enriched in His anticodons, as previous L: -His sites also were. Accordingly, these data add to existing experimental evidence for a stereochemical genetic code. In these peptide sites, bound amino acids approach each other to a proximity that allows a covalent peptide linkage. Isolation of several RNAs embracing two amino acids with a linking peptide bond supports the idea that a direct-RNA-template could encode primordial peptides, though crucial experiments remain.     

Binding of Human GM-CSF to Synthetic Peptides of the Alpha Subunit of Its Receptor

Abstract

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor α subunit (hGM-CSFRα). The sequences were chosen according to the homology between hGM-CSFRα and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. Four peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFRα and GHR was confirmed.     

The PK Domain of the Large Subunit of Herpes Simplex Virus Type 2 Ribonucleotide Reductase (ICP10) Is Required for Immediate-Early Gene Expression and Virus Growth

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10ΔPK). ICP10ΔPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10ΔPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10ΔPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.     

Identification of an HSV-1/HSV-2 cross-reactive T cell determinant.

The results from a number of studies have documented that the HSV glycoprotein gD is an important target for neutralizing antibodies. In contrast, little is known about the Th cell determinants present on HSV that are required for anti HSV gD antibody production. In our study we have immunized BALB/c mice with a recombinant source of HSV-1 gD lacking the carboxyl-terminal 93 amino acids. T cell hybridomas produced from the immunized animals recognized a single antigenic peptide (amino acids 246-261) in the context of I-Ad. The determinant expressed by gD peptide 246-261 was generated and presented by both HSV-1 and HSV-2 infected APC. Fine specificity analysis using truncated synthetic gD peptides revealed that the minimal amino acids recognized by the T hybrids were identical between HSV-1 and HSV-2. In addition, the minimal peptide-I-Ad binding analysis demonstrated that the minimal peptide sequence required for the binding to I-Ad and for T cell recognition contained two prolines. Thus, this important HSV antigenic determinant would not be expected to form an amphipathic alpha-helix and could therefore be missed by algorithms currently used to predict which amino acid sequences would be antigenic based on the propensity to form helices.     

Contamination Risks in Work with Synthetic Peptides: flg22 as an Example of a Pirate in Commercial Peptide Preparations

The pattern recognition receptor FLAGELLIN SENSING2 (FLS2) renders plant cells responsive to subnanomolar concentrations of flg22, the active epitope of bacterial flagellin. We recently observed that a preparation of the peptide IDL1, a signal known to regulate abscission processes via the receptor kinases HAESA and HAESA-like2, apparently triggered Arabidopsis thaliana cells in an FLS2-dependent manner. However, closer investigation revealed that this activity was due to contamination by a flg22-type peptide, and newly synthesized IDL1 peptide was completely inactive in FLS2 signaling. This raised alert over contamination events occurring in the process of synthesis or handling of peptides. Two recent reports have suggested that FLS2 has further specificities for structurally unrelated peptides derived from CLV3 and from Ax21. We thus scrutinized these peptides for activity in Arabidopsis cells as well. While responding to <1 nM flg22, Arabidopsis cells proved blind even to 100 μM concentrations of CLV3p and axY^s22. Our results confirm the exquisite sensitivity and selectivity of FLS2 for flg22. They also show that inadvertent contaminations with flg22-type peptides do occur and can be detected even in trace amounts by FLS2.During the last years, the pattern recognition receptor FLAGELLIN SENSING2 (FLS2) and its cognate microbe-associated molecular pattern (MAMP), the peptide flg22 (Felix et al., 1999), have widely been used to study plant innate immunity (Boller and Felix, 2009). Typically, in FLS2-expressing Arabidopsis thaliana cells, flg22 stimulates rapid changes of ion fluxes, including extracellular alkalinization and an induction of defense-related genes, such as FRK1, at threshold concentrations of 10 to 100 pM, while fls2 mutants lacking the receptor kinase FLS2 are completely unresponsive to flg22 (Boller and Felix, 2009). These findings demonstrate that FLS2 has an exquisite sensitivity as a flagellin receptor and that FLS2 is the only receptor for the flg22 ligand in Arabidopsis. Our previous results also indicated an exquisite selectivity of FLS2 with regard to its ligand. For example, the flg22 peptide of Agrobacterium tumefaciens (flg22^A.tum.) is completely inactive as a ligand of FLS2 or as a stimulus for FLS2-dependent responses and therefore has often been used as a negative control in assays for flg22-induced responses (Felix et al., 1999; Asai et al., 2002).Recently, we observed that a synthetic preparation of IDL1, an endogenous peptide signal involved in abscission processes (Stenvik et al., 2008), showed considerable activity as an inducer of MAMP responses and stimulated extracellular alkalinization in Arabidopsis cells at a threshold level of <5 nM (Figure 1A). More surprisingly, when examined in a cell culture of the fls2 efr double mutant, no significant medium alkalinization was detectable after treatment with the IDL1 preparation (Figure 1B). To check if FLS2 was involved in the response to the IDL1 preparation, we made use of the inhibitor flg22-Δ2, which acts as a specific antagonist of flg22 in Arabidopsis (Bauer et al., 2001). Indeed, presence of 30 μM flg22-Δ2 completely abolished the response to 50 nM IDL1 (Figure 1C).Open in a separate windowFigure 1.Effects of IDL1 Peptides on Extracellular pH in Suspension-Cultured Arabidopsis Cells.(A) Alkalinization in response to different doses of two independent preparations of the IDL1 peptide (preparations I and II). wt, wild-type.(B) Alkalinization response in cells from the fls2 efr double mutant.(C) Alkalinization response in wild-type cells to preparation I of IDL1 alone, to preparation I in combination with the flg22 antagonist flg22-Δ2 (30 μM), or to preparation I after digestion (overnight, 37°C) with endoproteinase AspN, as indicated.Based on strong genetic evidence, IDL1 is thought to act as a regulator of abscission processes via the receptor kinases HAESA and HAESA-like 2 (Stenvik et al., 2008), but why should FLS2 be involved? The IDL1 preparation was ∼100-fold less effective than authentic flg22 preparations, as indicated by the EC₅₀ values of 0.1 nM for flg22 (Bauer et al., 2001) and 10 nM for IDL1 (Figure 1A), respectively. We hypothesized that the IDL1 preparation might be contaminated by a peptide of the flg22 type. In contrast with IDL1, which has no acidic amino acid residues, flg22 contains two Asp residues that are important for its biological activity on FLS2 (Felix et al., 1999). We used this difference for selective digestion by the endoproteinase AspN, which cuts peptides N-terminal of Asp residues. Indeed, no activity was left in IDL1 after digestion (Figure 1C). This strongly indicated that the activity was not associated with the IDL1 peptide itself but rather with a flg22-type of contamination. Repurification of IDL1 by C18 reverse-phase chromatography could not separate the IDL1 peptide from the flg22 type of activity. Apart from a dominating signal for IDL1, mass spectrometry analysis of this fraction also revealed faint mass signatures characteristic for flg22 and its spontaneous derivative containing pyroglutamate at its N terminus (see Supplemental Figure 1 online). Together, these results clearly pinpointed a contamination as the source of the flg22-like activity in IDL1.Where did this contamination occur? We observed the same activity with a second, unopened tube from the same batch of the synthetic IDL1 peptide, indicating that a putative flg22 contamination had occurred prior to arrival in our lab, most likely in the company providing the peptide. We therefore resynthesized a new batch of IDL1 and found that it did not cause alkalinization in Arabidopsis wild-type cells even at a concentration of 10 μM (preparation II, Figure 1A). A further, independent, batch of an IDL1-derived peptide with a C-terminal extension by two amino acid residues similarly failed to induce alkalinization in the Arabidopsis wild-type cells (data not shown). These results made us acutely aware of a potential contamination problem when working with peptides unrelated to flg22. Indeed, two recent reports have suggested that FLS2 perceives two additional, unrelated, peptidic signals derived from either CLV3 (Lee et al., 2011) or Ax21 (Danna et al., 2011), respectively. What if these unexpected results were due to inadvertent contamination by flg22 as well?In a recent study (Mueller et al., 2012), we compared the FLS2 orthologs from Arabidopsis and tomato (Solanum lycopersicum) and their chimeras, making use of protoplasts from fls2 mutant plants transformed simultaneously with constructs encoding one of the FLS2 orthologs and a pFRK1:luciferase reporter, an assay system originally introduced by Asai et al. (2002). Protoplasts with both versions of FLS2 exhibited exquisite sensitivity to picomolar concentrations of flg22. However, they failed to respond to the hydroxylated CLV3 peptides CLV3-ΔAra₃-ΔH (12 amino acids) and CLV3-ΔAra₃ (13 amino acids) (described in Ohyama et al., 2009), termed CLV3p and CLV3p-H in our article (see Figure 1 in Mueller et al., 2012). Indeed, even when applied at a concentration of 100 μM, the 12–amino acid CLV3p caused no significant response in protoplasts expressing FLS2 from Arabidopsis (Figure 2A). A marginal transient increase in luminescence occurred in the first 2 h of the experiment, but this effect was also seen in the absence of FLS2 (Figure 3B), demonstrating that it had nothing to do with FLS2-dependent activation of the reporter gene. Our preparation of the CLV3p peptide exhibited the expected strong inhibitory effect in root growth assays with wild-type Arabidopsis and with fls2 mutants but not with the mutant clv2-1 (see Supplemental Figure 2 online).Open in a separate windowFigure 2.The CLV3p Peptide (Arg-Thr-Val-Hyp-Ser-Gly-Hyp-Asp-Pro-Leu-His-His, CLV3-ΔAra₃-ΔH in Ohyama et al., 2009) Does Not Induce Expression of the Reporter pFRK1:luciferase via the Receptor FLS2.Mesophyll protoplasts from efr×fls2 mutants were transformed with pFRK1:luciferase (pFRK1, promoter of the flagellin responsive receptor kinase 1) together with p35S:FLS2-GFP (A) or p35S:GFP (B) and tested for responsiveness to CLV3p and flg22 as indicated. GFP, green fluorescent protein; RLU, relative light units.Open in a separate windowFigure 3.Ax21-Derived Peptides axY^s22 and axY22A Show No Activity as Inducers of Oxidative Burst and Medium Alkalinization in Arabidopsis.(A) Oxidative burst in leaf pieces of Arabidopsis treated with axY^s22, axY22A, or flg22 as indicated. Reactive oxygen species (ROS) were determined by light emission (relative light units [RLU] of the luminometer) in a luminol-based assay. Values and error bars represent mean ± se of n = 6 replicates. (Error bars in all samples not treated with flg22 were smaller than 100 relative light units.)(B) Extracellular alkalinization in cell cultures of Arabidopsis treated with axY^s22, axY22A, or a control peptide (SASRSRIQDADFAAETANLSRSQILQQAGTA) in combination with flg22, as indicated.In previous work by Lee et al. (2009), the sulfated peptide axY^s22, derived from the protein Ax21 of the pathogenic bacterium Xanthomonas oryzae, but not its variant form axY22A, in which the sulfotyrosine was replaced by an Ala, have been reported to cause a resistance response in rice (Oryza sativa) expressing the receptor kinase XA21. Recently, preparations of both of these two peptides have been reported to stimulate immune responses in Arabidopsis when applied at concentrations of 1 to 100 μM (Danna et al., 2011; Figures 1 to 3). Surprisingly, this activation was dependent on the presence of a functional FLS2 receptor, suggesting that these peptides are acting as ligands for FLS2 as well. We obtained fresh preparations of both axY^s22 and axY22A. In our hands, both peptides were completely inactive at concentrations up to 100 μM in oxidative burst and alkalinization assays (Figure 3). The cells used for the alkalinization assays strongly responded to 100 pM of authentic flg22, indicating that the Ax21-related peptides were at least a million times less efficient to induce alkalinization via FLS2 (Figure 3B).The peptide flg22-Δ2 functions as a competitive antagonist that specifically inhibits flg22-induced responses in Arabidopsis (Bauer et al., 2001). Since the FLS2-dependent responses to CLV3p and the Ax21 peptides were reported to be inhibited by excess flg22-Δ2 (Danna et al., 2011; Lee et al., 2011), we also checked whether CLV3p or the Ax21 peptides might interfere with the binding of flg22 to FLS2 (Figure 4). The receptor FLS2 binds carrier-free ¹²⁵I-Tyr-flg22 with a high affinity, and this binding can be specifically competed by 10 μM unlabeled flg22 but neither by 30 μM CLV3p nor by 30 μM axY^s22 (Figure 4). Thus, we cannot confirm that CLV3p or axY^s22 can directly interact and activate FLS2. While inadvertent contamination is a possible explanation, we cannot finally explain the obvious discrepancies to the results in the Lee et al. (2011) and Danna et al. (2011) reports. Also, since our analysis focused on direct and immediate effects on FLS2, we cannot comment on effects that high concentrations of peptides like CLV3p or axY^s22 might exert on prolonged treatment. For example, induction of plant resistance is a highly complex process that develops over days and involves two living systems. Rather than on the host cells, peptides applied might act on the pathogenic bacteria and influence their synthesis of flagellin or their assembly/disassembly of flagellin subunits into flagellar structures. At least for the ax21 peptides, described as a quorum sensing type of signals for bacteria, this is an option to be considered.Open in a separate windowFigure 4.The Peptides axY^s22 and CLV3p Do Not Compete for Binding of flg22 to FLS2.Binding of ¹²⁵I-Tyr-flg22 to wild-type Arabidopsis seedlings in the absence of competitor, in the presence of 10 μM unlabeled flg22, or in the presence of 30 μM unlabeled axY^s22 or CLV3p. Bars and error bars represent radioactivity (counts per min [cpm]) bound to plant material as means and sd of n = 3 replicates.Our results confirm the exquisite sensitivity and selectivity of FLS2 for its cognate ligand, flg22. They also show that extreme care must be taken when attempting to assess the effect of peptides on responses that can also be elicited by flg22. Based on our experience, peptide preparations ordered from different commercial suppliers may occasionally be contaminated by flg22-related activity. We observed a contamination corresponding to ∼1% of flg22 equivalents in one of the IDL1 preparations (Figure 1). However, we would like to emphasize that in a peptide preparation applied at 100 μM a contamination by flg22 of only ∼0.0001% (∼1 ppm) can activate FLS2-dependent responses. Using HPLC and mass spectrometry analysis as common checks for purity, suppliers guarantee that a certain percentage, maximally 99%, of the preparation corresponds to the peptide ordered. However, as exemplified for the contaminated IDL1 preparation (see Supplemental Figure 1 online), contaminations at or below 1% can easily go unnoticed. Also, normally, the molecular characteristics of a potential contaminant are not known, so a flg22-type of activity could be present as a partial degradation product or in the form of an unknown flg22 derivative.We cannot estimate a frequency for cross-contaminations in peptide preparations, but it seems to occur surprisingly often. Over the years, we ordered >100 peptides from various commercial suppliers and had at least two further incidents with flg22-type contaminations. In one of these cases, we ordered, and obtained, flg22 and three structurally unrelated peptides. We found residual flg22-type activity in two out of the three preparations of these unrelated peptides, indicating contamination in the course of commercial peptide synthesis (in this case, by a supplier different from the provider of IDL1) or during handling of these peptides in our lab.There are reasons why contaminations by flg22 might pose a particular risk. First, flg22 has a tendency to stick to surfaces and we recommended the use of 0.1 M NaCl and 1 mg/mL BSA to prevent loss of the peptide during serial dilutions (Felix et al., 1999). In turn, flg22 adhering to tubings, columns, or glassware might provide a source of contamination for peptides getting handled subsequently. Second, we noticed that lyophilized flg22 can easily pick up electrostatic charge and is prone to float around with the slightest streams of air. This could be a particular problem also for preparations handled by robots of peptide manufacturers. Third, due to a considerable demand by an increasing number of labs, flg22-related peptides have been ordered from various peptide suppliers numerous times and, picking up this peptide as inadvertent contamination has become a considerable problem.In conclusion, our study complements and extends the commentary by Segonzac et al. (2012) by demonstrating that the receptor FLS2 has an extraordinarily high affinity and selectivity to its ligand, flg22, and that it is completely blind to the peptides IDL1, CLV3p, axY^s22, and axY22A even in our most sensitive bioassays. Our results and arguments do not apodictically exclude that a receptor like FLS2 could have a second, physiologically relevant, ligand. Also, there may be chemical structures that inadvertently act as surrogates or mimetics of the true ligand flg22. However, in view of the high selectivity of the FLS2 for its genuine ligand flg22, we think the probability of alien interactors is rather low. By contrast, contaminations with flg22-related molecules can and do occur.How can contamination of bioactive peptides be recognized and avoided? The first, probably most important, point is a sharpened awareness about in-lab and supplier-dependent sources of contamination. These risks are often ignored, in particular when working with synthetic, purified peptides. A purity of >95 or >99%, as guaranteed by suppliers, is of limited value with respect to highly active contaminants detectable even at the ppm level. Purification offered by suppliers certainly helps to remove chemicals used in the synthesis process and to get rid of many incomplete variants of the peptide ordered. However, at least theoretically, contaminated equipment used during the purification might contribute to the problem rather than to its solution. Dose–response relationships for the peptides under scrutiny are important to consider physiological relevance in general and to compare activities with published data in particular. Thereby, the higher the dose of a peptide applied, the higher the risk to pick up even spurious contaminants. Furthermore, analysis of bioactive peptides should not depend on a single peptide preparation alone. Peptide variants are crucial to elucidate the specificity of an interaction process. The use of several, independently synthesized and handled peptide preparations should help to reliably detect sporadic contamination events and to distinguish contaminants from true ligands. Finally, at least for the particular problem of contamination by flg22, we can offer testing peptide preparations using the sensitive bioassays established in our labs. As long as we have the hands and capacity to handle such requests, we certainly would like to contribute with such a service to detect pirate peptides.     

Disruption of Rhodopsin Dimerization with Synthetic Peptides Targeting an Interaction Interface

Although homo- and heterodimerizations of G protein-coupled receptors (GPCRs) are well documented, GPCR monomers are able to assemble in different ways, thus causing variations in the interactive interface between receptor monomers among different GPCRs. Moreover, the functional consequences of this phenomenon, which remain to be clarified, could be specific for different GPCRs. Synthetic peptides derived from transmembrane (TM) domains can interact with a full-length GPCR, blocking dimer formation and affecting its function. Here we used peptides corresponding to TM helices of bovine rhodopsin (Rho) to investigate the Rho dimer interface and functional consequences of its disruption. Incubation of Rho with TM1, TM2, TM4, and TM5 peptides in rod outer segment (ROS) membranes shifted the resulting detergent-solubilized protein migration through a gel filtration column toward smaller molecular masses with a reduced propensity for dimer formation in a cross-linking reaction. Binding of these TM peptides to Rho was characterized by both mass spectrometry and a label-free assay from which dissociation constants were calculated. A BRET (bioluminescence resonance energy transfer) assay revealed that the physical interaction between Rho molecules expressed in membranes of living cells was blocked by the same four TM peptides identified in our in vitro experiments. Although disruption of the Rho dimer/oligomer had no effect on the rates of G protein activation, binding of G_t to the activated receptor stabilized the dimer. However, TM peptide-induced disruption of dimer/oligomer decreased receptor stability, suggesting that Rho supramolecular organization could be essential for ROS stabilization and receptor trafficking.