Biolistic transformation of the yeast Saccharomyces cerevisiae mitochondrial DNA.

The insertion of genes into mitochondria by biolistic transformation is currently only possible in the yeast Saccharomyces cerevisiae and the algae Chlamydomonas reinhardtii. The fact that S. cerevisiae mitochondria can exist with partial (ρ- mutants) or complete deletions (ρ0 mutants) of mitochondrial DNA (mtDNA), without requiring a specific origin of replication, enables the propagation of exogenous sequences. Additionally, mtDNA in this organism undergoes efficient homologous recombination, making it well-suited for genetic manipulation. In this review, we present a summarized historical overview of the development of biolistic transformation and discuss iconic applications of the technique. We also provide a detailed example on how to obtain transformants with recombined foreign DNA in their mitochondrial genome.

Overexpression of MRX9 impairs processing of RNAs encoding mitochondrial oxidative phosphorylation factors COB and COX1 in yeast.

Mitochondrial translation is a highly regulated process, and newly synthesized mitochondrial products must first associate with several nuclear-encoded auxiliary factors to form oxidative phosphorylation complexes. The output of mitochondrial products should therefore be in stoichiometric equilibrium with the nuclear-encoded products to prevent unnecessary energy expense or the accumulation of pro-oxidant assembly modules. In the mitochondrial DNA of Saccharomyces cerevisiae, COX1 encodes subunit 1 of the cytochrome c oxidase and COB the central core of the cytochrome bc1 electron transfer complex; however, factors regulating the expression of these mitochondrial products are not completely described. Here, we identified Mrx9p as a new factor that controls COX1 and COB expression. We isolated MRX9 in a screen for mitochondrial factors that cause poor accumulation of newly synthesized Cox1p and compromised transition to the respiratory metabolism. Northern analyses indicated lower levels of COX1 and COB mature mRNAs accompanied by an accumulation of unprocessed transcripts in the presence of excess Mrx9p. In a strain devoid of mitochondrial introns, MRX9 overexpression did not affect COX1 and COB translation or respiratory adaptation, implying Mrx9p regulates processing of COX1 and COB RNAs. In addition, we found Mrx9p was localized in the mitochondrial inner membrane, facing the matrix, as a portion of it cosedimented with mitoribosome subunits and its removal or overexpression altered Mss51p sedimentation. Finally, we showed accumulation of newly synthesized Cox1p in the absence of Mrx9p was diminished in cox14 null mutants. Taken together, these data indicate a regulatory role of Mrx9p in COX1 RNA processing.

Functional analyses of mitoribosome 54S subunit devoid of mitochondria-specific protein sequences.

In Saccharomyces cerevisiae, mitoribosomes are composed of a 54S large subunit (mtLSU) and a 37S small subunit (mtSSU). The two subunits altogether contain 73 mitoribosome proteins (MRPs) and two ribosomal RNAs (rRNAs). Although mitoribosomes preserve some similarities with their bacterial counterparts, they have significantly diverged by acquiring new proteins, protein extensions, and new RNA segments, adapting the mitoribosome to the synthesis of highly hydrophobic membrane proteins. In this study, we investigated the functional relevance of mitochondria-specific protein extensions at the C-terminus (C) or N-terminus (N) present in 19 proteins of the mtLSU. The studied mitochondria-specific extensions consist of long tails and loops extending from globular domains that mainly interact with mitochondria-specific proteins and 21S rRNA moieties extensions. The expression of variants devoid of extensions in uL4 (C), uL5 (N), uL13 (N), uL13 (C), uL16 (C), bL17 (N), bL17 (C), bL21 (24), uL22 (N), uL23 (N), uL23 (C), uL24 (C), bL27 (C), bL28 (N), bL28 (C), uL29 (N), uL29 (C), uL30 (C), bL31 (C), and bL32 (C) did not rescue the mitochondrial protein synthesis capacities and respiratory growth of the respective null mutants. On the contrary, the truncated form of the mitoribosome exit tunnel protein uL24 (N) yields a partially functional mitoribosome. Also, the removal of mitochondria-specific sequences from uL1 (N), uL3 (N), uL16 (N), bL9 (N), bL19 (C), uL29 (C), and bL31 (N) did not affect the mitoribosome function and respiratory growth. The collection of mutants described here provides new means to study and evaluate defective assembly modules in the mitoribosome biogenesis process.

Coq3p relevant residues for protein activity and stability.

Coenzyme Q (CoQ) is an essential molecule that consists of a highly substituted benzene ring attached to a polyprenyl tail anchored in the inner mitochondrial membrane. CoQ transfers electrons from NADH dehydrogenase and succinate dehydrogenase complexes toward ubiquinol-cytochrome c reductase, and that allows aerobic growth of cells. In Saccharomyces cerevisiae, the synthesis of CoQ depends on fourteen proteins Coq1p-Co11p, Yah1p, Arh1p, and Hfd1p. Some of these proteins are components of CoQ synthome. Using ab initio molecular modeling and site-directed mutagenesis, we identified the functional residues of the O-methyltransferase Coq3p, which depends on S-adenosylmethionine for catalysis and is necessary for two O-methylation steps required for CoQ maturation. Conserved residues as well as those that coevolved in the protein structure were found to have important roles in respiratory growth, CoQ biosynthesis, and also in the stability of CoQ synthome proteins. Finally, a multiple sequence alignment showed that S. cerevisiae Coq3p has a 45 amino acid residues insertion that is poorly conserved or absent in oleaginous yeast, cells that can store up to 20% of their dry weight as lipids. These results point to the Coq3p structural determinants of its biological and catalytic function and could contribute to the development of lipid-producing yeast for biotechnology.

Human Mitochondrial Pathologies of the Respiratory Chain and ATP Synthase: Contributions from Studies of Saccharomyces cerevisiae.

The ease with which the unicellular yeast Saccharomyces cerevisiae can be manipulated genetically and biochemically has established this organism as a good model for the study of human mitochondrial diseases. The combined use of biochemical and molecular genetic tools has been instrumental in elucidating the functions of numerous yeast nuclear gene products with human homologs that affect a large number of metabolic and biological processes, including those housed in mitochondria. These include structural and catalytic subunits of enzymes and protein factors that impinge on the biogenesis of the respiratory chain. This article will review what is currently known about the genetics and clinical phenotypes of mitochondrial diseases of the respiratory chain and ATP synthase, with special emphasis on the contribution of information gained from pet mutants with mutations in nuclear genes that impair mitochondrial respiration. Our intent is to provide the yeast mitochondrial specialist with basic knowledge of human mitochondrial pathologies and the human specialist with information on how genes that directly and indirectly affect respiration were identified and characterized in yeast.

The translational activator Sov1 coordinates mitochondrial gene expression with mitoribosome biogenesis.

Mitoribosome biogenesis is an expensive metabolic process that is essential to maintain cellular respiratory capacity and requires the stoichiometric accumulation of rRNAs and proteins encoded in two distinct genomes. In yeast, the ribosomal protein Var1, alias uS3m, is mitochondrion-encoded. uS3m is a protein universally present in all ribosomes, where it forms part of the small subunit (SSU) mRNA entry channel and plays a pivotal role in ribosome loading onto the mRNA. However, despite its critical functional role, very little is known concerning VAR1 gene expression. Here, we demonstrate that the protein Sov1 is an in bona fide VAR1 mRNA translational activator and additionally interacts with newly synthesized Var1 polypeptide. Moreover, we show that Sov1 assists the late steps of mtSSU biogenesis involving the incorporation of Var1, an event necessary for uS14 and mS46 assembly. Notably, we have uncovered a translational regulatory mechanism by which Sov1 fine-tunes Var1 synthesis with its assembly into the mitoribosome.

COQ11 deletion mitigates respiratory deficiency caused by mutations in the gene encoding the coenzyme Q chaperone protein Coq10.

Coenzyme Q (Q n ) is a vital lipid component of the electron transport chain that functions in cellular energy metabolism and as a membrane antioxidant. In the yeast Saccharomyces cerevisiae, coq1-coq9 deletion mutants are respiratory-incompetent, sensitive to lipid peroxidation stress, and unable to synthesize Q6 The yeast coq10 deletion mutant is also respiratory-deficient and sensitive to lipid peroxidation, yet it continues to produce Q6 at an impaired rate. Thus, Coq10 is required for the function of Q6 in respiration and as an antioxidant and is believed to chaperone Q6 from its site of synthesis to the respiratory complexes. In several fungi, Coq10 is encoded as a fusion polypeptide with Coq11, a recently identified protein of unknown function required for efficient Q6 biosynthesis. Because "fused" proteins are often involved in similar biochemical pathways, here we examined the putative functional relationship between Coq10 and Coq11 in yeast. We used plate growth and Seahorse assays and LC-MS/MS analysis to show that COQ11 deletion rescues respiratory deficiency, sensitivity to lipid peroxidation, and decreased Q6 biosynthesis of the coq10Δ mutant. Additionally, immunoblotting indicated that yeast coq11Δ mutants accumulate increased amounts of certain Coq polypeptides and display a stabilized CoQ synthome. These effects suggest that Coq11 modulates Q6 biosynthesis and that its absence increases mitochondrial Q6 content in the coq10Δcoq11Δ double mutant. This augmented mitochondrial Q6 content counteracts the respiratory deficiency and lipid peroxidation sensitivity phenotypes of the coq10Δ mutant. This study further clarifies the intricate connection between Q6 biosynthesis, trafficking, and function in mitochondrial metabolism.

Modular biogenesis of mitochondrial respiratory complexes.

Mitochondrial function relies on the activity of oxidative phosphorylation to synthesise ATP and generate an electrochemical gradient across the inner mitochondrial membrane. These coupled processes are mediated by five multi-subunit complexes that reside in this inner membrane. These complexes are the product of both nuclear and mitochondrial gene products. Defects in the function or assembly of these complexes can lead to mitochondrial diseases due to deficits in energy production and mitochondrial functions. Appropriate biogenesis and function are mediated by a complex number of assembly factors that promote maturation of specific complex subunits to form the active oxidative phosphorylation complex. The understanding of the biogenesis of each complex has been informed by studies in both simple eukaryotes such as Saccharomyces cerevisiae and human patients with mitochondrial diseases. These studies reveal each complex assembles through a pathway using specific subunits and assembly factors to form kinetically distinct but related assembly modules. The current understanding of these complexes has embraced the revolutions in genomics and proteomics to further our knowledge on the impact of mitochondrial biology in genetics, medicine, and evolution.

Mutations of Cys and Ser residues in the α5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan.

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the α5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (α5-C76S or α5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that α5-C76S or α5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random α5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., α-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the α5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the α5-subunit, and consequently impacts the lifespan of yeast.

5' processing of Saccharomyces cerevisiae mitochondrial tRNAs requires expression of multiple genes.

Mitochondrial tRNAs are processed at their 5'ends by highly divergent but ubiquitous RNase P. In Saccharomyces cerevisiae, Rpm2p is the protein component of RNase P. Here, we identify four novel genes MTA1, MTA2, GEP5 and PET130 of the Saccharomycetaceae family that are necessary for an efficient processing of mitochondrial tRNAs. Null mutants of mta1, mta2 and gep5 have severely reduced levels of mitochondrial tRNAs; in addition, temperature sensitive (ts) mutants of mta1, mta2, pet130 and gep5 accumulated tRNAs precursor transcripts at the restrictive but not at the permissive temperature. The same mitochondrial tRNAs precursors were also identified in rpm2 ts mutants or in the double ts mutants mta1 rpm2 and mta2 rpm2. The genetic and physical association of these four novel genes corroborate the hypothesis that they have their function associated. Different combinations of mta1, mta2, pet130 and gep5 ts alleles display a synthetic respiratory deficient phenotype, an indication of genetic interactions of the genes. Indeed, Mta1p, Mta2p, Pet130p, and Gep5p are associated with the mitochondrial inner membrane and are all extracted and sediment in sucrose gradients as high molecular weight complexes, where they may be present in a common complex with Rpm2p. This is supported by pull-down assays showing co-immunopurification of Rpm2 with Mta1p.

Msc6p is required for mitochondrial translation initiation in the absence of formylated Met-tRNAfMet.

Mitochondrial translation normally requires formylation of the initiator tRNA-met, a reaction catalyzed by the enzyme formyltransferase, Fmt1p and MTFMT in Saccharomyces cerevisiae and human mitochondria, respectively. Yeast fmt1 mutants devoid of Fmt1p, however, can synthesize all mitochondrial gene products by initiating translation with a non-formylated methionyl-tRNA. Yeast synthetic respiratory-deficient fmt1 mutants have uncovered several factors suggested to play a role in translation initiation with non-formylated methionyl-tRNA. Here, we present evidence that Msc6p, a member of the pentatricopeptide repeat (PPR) motif family, is another essential factor for mitochondrial translation in fmt1 mutants. The PPR motif is characteristic of RNA-binding proteins found in chloroplasts and plant and fungal mitochondria, and is generally involved in RNA stability and transport. Moreover, in the present study, we show that the respiratory deficiency of fmt1msc6 double mutants can be rescued by overexpression of the yeast mitochondrial initiation factor mIF-2, encoded by IFM1. The role of Msc6p in translational initiation is further supported by pull-down assays showing that it transiently interacts with mIF-2. Altogether, our data indicate that Msc6p is an important factor in mitochondrial translation with an auxiliary function related to the mIF-2-dependent formation of the initiation complex.

Mutations of Cys and Ser residues in the alpha5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the alpha5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (alpha5-C76S or alpha5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that alpha5-C76S or alpha5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random alpha5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., a-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the alpha5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the a5-subunit, and consequently impacts the lifespan of yeast.

Mutations of Cys and Ser residues in the alpha5-subunit of the 20S proteasome from Saccharomyces cerevisiae affects gating and chronological lifespan

In addition to autophagy, proteasomes are critical for regulating intracellular protein levels and removing misfolded proteins. The 20S proteasome (20SPT), the central catalytic unit, is sometimes flanked by regulatory units at one or both ends. Additionally, proteosomal activation has been associated with increased lifespan in many organisms. Our group previously reported that the gating (open/closed) of the free 20S proteasome is redox controlled, and that S-glutathionylation of two Cys residues (Cys76 and Cys221) in the alpha5 subunit promotes gate opening. The present study constructed site-directed mutants of these Cys residues, and evaluated the effects these mutations have on proteosome gate opening and yeast cell survival. Notably, the double mutation of both Cys residues (Cys76 and Cys221) rendered the cells nonviable, whereas the lifespan of the yeast carrying the single mutations (alpha5-C76S or alpha5-C221S) was attenuated when compared to the wild type counterpart. Furthermore, it was found that alpha5-C76S or alpha5-C221S 20SPT were more likely to be found with the gate in a closed conformation. In contrast, a random alpha5-subunit double mutation (S35P/C221S) promoted gate opening, increased chronological lifespan and provided resistance to oxidative stress. The 20SPT core particle purified from the long-lived strain degraded model proteins (e.g., a-synuclein) more efficiently than preparations obtained from the wild-type counterpart, and also displayed an increased chymotrypsin-like activity. Mass spectrometric analyses of the C76S, C221S, S35P/C221S, S35P and S35P/C76S mutants provided evidence that the highly conserved Cys76 residue of the alpha5-subunit is the key determinant for gate opening and cellular survival. The present study reveals a sophisticated regulatory mechanism that controls gate opening, which appears to be based on the interactions among multiple residues within the a5-subunit, and consequently impacts the lifespan of yeast.

Metabolic studies of a patient harbouring a novel S487L mutation in the catalytic subunit of AMPK.

AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.

Mitochondrial ribosome bL34 mutants present diminished translation of cytochrome c oxidase subunits.

Saccharomyces cerevisiae mitoribosomes are specialized in the translation of a few number of highly hydrophobic membrane proteins, components of the oxidative phosphorylation system. Mitochondrial characteristics, such as the membrane system and its redox state driven mitoribosomes evolution through great diversion from their bacterial and cytosolic counterparts. Therefore, mitoribosome presents a considerable number of mitochondrial-specific proteins, as well as new protein extensions. In this work we characterize temperature sensitive mutants of the subunit bL34 present in the 54S large subunit. Although bL34 has bacterial homologs, in yeast it has a long 65 aminoacids mitochondrial N-terminal addressing sequence, here we demonstrate that it can be replaced by the mitochondrial addressing sequence of Neurospora crassa ATP9 gene. The bL34 temperature sensitive mutants present lowered translation of mitochondrial COX1 and COX3, which resulted in reduced cytochrome c oxidase activity and respiratory growth deficiency. The sedimentation properties of bL34 in sucrose gradients suggest that similarly to its bacterial homolog, bL34 is also a later participant in the process of mitoribosome biogenesis.

Melatonin improves survival and respiratory activity of yeast cells challenged by alpha-synuclein and menadione.

One of the hallmarks of Parkinson disease is α-synuclein aggregate deposition that leads to endoplasmic reticulum stress, Golgi fragmentation and impaired energy metabolism with consequent redox imbalance. In the last decade, many studies have used Saccharomyces cerevisiae as a model in order to explore the intracellular consequences of α-synuclein overexpression. In this study we propose to evaluate the respiratory outcome of yeast cells expressing α-synuclein. Cell viability or growth on selective media for respiratory activity was mainly affected in the α-synuclein-expressing cells if they were also treated with menadione, which stimulates reactive oxygen species production. We also tested whether melatonin, a natural antioxidant, would counteract the deleterious effects of α-synuclein and menadione. In fact, melatonin addition improved the respiratory growth of α-synuclein/menadione-challenged cells, presented a general improvement in the enzymatic activity of the respiratory complexes and finally elevated the rate of mitophagy, an important cellular process necessary for the clearance of damaged mitochondria. Altogether, our data confirms that α-synuclein impairs respiration in yeast, which can be rescued by melatonin addition.

Proteolytic cleavage by the inner membrane peptidase (IMP) complex or Oct1 peptidase controls the localization of the yeast peroxiredoxin Prx1 to distinct mitochondrial compartments.

Yeast Prx1 is a mitochondrial 1-Cys peroxiredoxin that catalyzes the reduction of endogenously generated H2O2 Prx1 is synthesized on cytosolic ribosomes as a preprotein with a cleavable N-terminal presequence that is the mitochondrial targeting signal, but the mechanisms underlying Prx1 distribution to distinct mitochondrial subcompartments are unknown. Here, we provide direct evidence of the following dual mitochondrial localization of Prx1: a soluble form in the intermembrane space and a form in the matrix weakly associated with the inner mitochondrial membrane. We show that Prx1 sorting into the intermembrane space likely involves the release of the protein precursor within the lipid bilayer of the inner membrane, followed by cleavage by the inner membrane peptidase. We also found that during its import into the matrix compartment, Prx1 is sequentially cleaved by mitochondrial processing peptidase and then by octapeptidyl aminopeptidase 1 (Oct1). Oct1 cleaved eight amino acid residues from the N-terminal region of Prx1 inside the matrix, without interfering with its peroxidase activity in vitro Remarkably, the processing of peroxiredoxin (Prx) proteins by Oct1 appears to be an evolutionarily conserved process because yeast Oct1 could cleave the human mitochondrial peroxiredoxin Prx3 when expressed in Saccharomyces cerevisiae Altogether, the processing of peroxiredoxins by Imp2 or Oct1 likely represents systems that control the localization of Prxs into distinct compartments and thereby contribute to various mitochondrial redox processes.

Aep3p-dependent translation of yeast mitochondrial ATP8.

Translation of mitochondrial gene products in Saccharomyces cerevisiae depends on mRNA-specific activators that bind to the 5' untranslated regions and promote translation on mitochondrial ribosomes. Here we find that Aep3p, previously shown to stabilize the bicistronic ATP8-ATP6 mRNA and facilitate initiation of translation from unformylated methionine, also activates specifically translation of ATP8 This is supported by several lines of evidence. Temperature-sensitive aep3 mutants are selectively blocked in incorporating [35S]methionine into Atp8p at nonpermissive but not at the permissive temperature. This phenotype is not a consequence of defective transcription or processing of the pre-mRNA. Neither is it explained by turnover of Aep3p, as evidenced by the failure of aep3 mutants to express a recoded ARG8m when this normally nuclear gene is substituted for ATP8 in mitochondrial DNA. Finally, translational of ATP8 mRNA in aep3 mutants is partially rescued by recoded allotopic ATP8 (nATP8) in a high-expression plasmid or in a CEN plasmid in the presence of recessive mutations in genes involved in stability and polyadenylation of RNA.

pRb and CyclinD1 Complement p16 as Immunohistochemical Surrogate Markers of HPV Infection in Head and Neck Cancer.

Identification of human papillomavirus (HPV) association in head and neck squamous cell carcinoma (HNSCC) is important to identify patients with favorable disease course. However, molecular HPV detection is not universally available. p16 has been proposed as a surrogate marker for HPV infection in HNSCC but, use on its own may result in wrong assignment of some cases to the group of HPV-associated tumors. We have therefore studied 424 HNSCC cases with known p16 and HPV DNA polymerase chain reaction (PCR) status for expression of retinoblastoma protein (pRb) and CyclinD1 by immunohistochemistry using 6-tiered scales (0 to 5) and a combined score (0 to 10). Sixty-one of 424 cases showed overexpression of p16. Of these, 52 cases were HPV DNA-PCR-positive. HPV association strongly correlated with low expression scores for pRb and CyclinD1 individually (scores ≤2) or combined (score sum ≤4), whereas HPV-negative carcinomas showed widely distributed expression scores. High expression scores for pRb or for pRb/CyclinD1 were observed exclusively in HPV DNA-PCR-negative cases. Three of 9 p16-positive/HPV DNA-PCR-negative cases showed high expression of pRb and displayed a high combined pRb/CyclinD1 score. We conclude that HPV-positive HNSCC are characterized by p16 overexpression and low scores for pRb, CyclinD1, and a low combined pRb/CyclinD1 score. High pRb or combined pRb/CyclinD1 scores are strong indicators for HPV-negativity and may justify excluding these cases from further molecular HPV testing. Furthermore p16-positive/HPV DNA-PCR-negative cases show heterogeneous expression of pRb and CyclinD1, including high pRb or high combined pRb/CyclinD1 scores suggesting that at least some of these cases are truly HPV negative.