Mechanisms and regulation of substrate degradation by the 26S proteasome.

The 26S proteasome is involved in degrading and regulating the majority of proteins in eukaryotic cells, which requires a sophisticated balance of specificity and promiscuity. In this Review, we discuss the principles that underly substrate recognition and ATP-dependent degradation by the proteasome. We focus on recent insights into the mechanisms of conventional ubiquitin-dependent and ubiquitin-independent protein turnover, and discuss the plethora of modulators for proteasome function, including substrate-delivering cofactors, ubiquitin ligases and deubiquitinases that enable the targeting of a highly diverse substrate pool. Furthermore, we summarize recent progress in our understanding of substrate processing upstream of the 26S proteasome by the p97 protein unfoldase. The advances in our knowledge of proteasome structure, function and regulation also inform new strategies for specific inhibition or harnessing the degradation capabilities of the proteasome for the treatment of human diseases, for instance, by using proteolysis targeting chimera molecules or molecular glues.

The 26S Proteasome Utilizes a Kinetic Gateway to Prioritize Substrate Degradation.

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.

Structure and Function of the 26S Proteasome.

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

The Ufd1 cofactor determines the linkage specificity of polyubiquitin chain engagement by the AAA+ ATPase Cdc48.

The AAA+ ATPase Cdc48 utilizes the cofactor Ufd1/Npl4 to bind and thread polyubiquitinated substrates for their extraction from complexes or membranes and often for subsequent proteasomal degradation. Previous studies indicated that Cdc48 engages polyubiquitin chains through the Npl4-mediated unfolding of an initiator ubiquitin; yet, the underlying principles remain largely unknown. Using FRET-based assays, we revealed the mechanisms and kinetics of ubiquitin unfolding, insertion into the ATPase, and unfolding of the ubiquitin-attached substrate. We found that Cdc48 uses Ufd1's UT3 domain to bind a K48-linked ubiquitin on the initiator's proximal side of the chain, thereby directing the initiator toward rapid unfolding by Npl4 and engagement by Cdc48. Ubiquitins on the initiator's distal side increase substrate affinity and facilitate unfolding but impede substrate release from Cdc48-Ufd1/Npl4 in the absence of additional cofactors. Our findings explain how Cdc48-UN efficiently processes substrates with K48-linked chains of 4-6 ubiquitins, which represent most cellular polyubiquitinated proteins.

The ClpXP protease unfolds substrates using a constant rate of pulling but different gears.

ATP-dependent proteases are vital to maintain cellular protein homeostasis. Here, we study the mechanisms of force generation and intersubunit coordination in the ClpXP protease from E. coli to understand how these machines couple ATP hydrolysis to mechanical protein unfolding. Single-molecule analyses reveal that phosphate release is the force-generating step in the ATP-hydrolysis cycle and that ClpXP translocates substrate polypeptides in bursts resulting from highly coordinated conformational changes in two to four ATPase subunits. ClpXP must use its maximum successive firing capacity of four subunits to unfold stable substrates like GFP. The average dwell duration between individual bursts of translocation is constant, regardless of the number of translocating subunits, implying that ClpXP operates with constant "rpm" but uses different "gears."

ClpX(P) generates mechanical force to unfold and translocate its protein substrates.

AAA(+) unfoldases denature and translocate polypeptides into associated peptidases. We report direct observations of mechanical, force-induced protein unfolding by the ClpX unfoldase from E. coli, alone, and in complex with the ClpP peptidase. ClpX hydrolyzes ATP to generate mechanical force and translocate polypeptides through its central pore. Threading is interrupted by pauses that are found to be off the main translocation pathway. ClpX's translocation velocity is force dependent, reaching a maximum of 80 aa/s near-zero force and vanishing at around 20 pN. ClpX takes 1, 2, or 3 nm steps, suggesting a fundamental step-size of 1 nm and a certain degree of intersubunit coordination. When ClpX encounters a folded protein, it either overcomes this mechanical barrier or slips on the polypeptide before making another unfolding attempt. Binding of ClpP decreases the slip probability and enhances the unfolding efficiency of ClpX. Under the action of ClpXP, GFP unravels cooperatively via a transient intermediate.

Mechanism for the Regulated Control of Bacterial Transcription Termination by a Universal Adaptor Protein.

NusG/Spt5 proteins are the only transcription factors utilized by all cellular organisms. In enterobacteria, NusG antagonizes the transcription termination activity of Rho, a hexameric helicase, during the synthesis of ribosomal and actively translated mRNAs. Paradoxically, NusG helps Rho act on untranslated transcripts, including non-canonical antisense RNAs and those arising from translational stress; how NusG fulfills these disparate functions is unknown. Here, we demonstrate that NusG activates Rho by assisting helicase isomerization from an open-ring, RNA-loading state to a closed-ring, catalytically active translocase. A crystal structure of closed-ring Rho in complex with NusG reveals the physical basis for this activation and further explains how Rho is excluded from translationally competent RNAs. This study demonstrates how a universally conserved transcription factor acts to modulate the activity of a ring-shaped ATPase motor and establishes how the innate sequence bias of a termination factor can be modulated to silence pervasive, aberrant transcription.

The N1 domain of the peroxisomal AAA-ATPase Pex6 is required for Pex15 binding and proper assembly with Pex1.

The heterohexameric ATPases associated with diverse cellular activities (AAA)-ATPase Pex1/Pex6 is essential for the formation and maintenance of peroxisomes. Pex1/Pex6, similar to other AAA-ATPases, uses the energy from ATP hydrolysis to mechanically thread substrate proteins through its central pore, thereby unfolding them. In related AAA-ATPase motors, substrates are recruited through binding to the motor's N-terminal domains or N terminally bound cofactors. Here, we use structural and biochemical techniques to characterize the function of the N1 domain in Pex6 from budding yeast, Saccharomyces cerevisiae. We found that although Pex1/ΔN1-Pex6 is an active ATPase in vitro, it does not support Pex1/Pex6 function at the peroxisome in vivo. An X-ray crystal structure of the isolated Pex6 N1 domain shows that the Pex6 N1 domain shares the same fold as the N-terminal domains of PEX1, CDC48, and NSF, despite poor sequence conservation. Integrating this structure with a cryo-EM reconstruction of Pex1/Pex6, AlphaFold2 predictions, and biochemical assays shows that Pex6 N1 mediates binding to both the peroxisomal membrane tether Pex15 and an extended loop from the D2 ATPase domain of Pex1 that influences Pex1/Pex6 heterohexamer stability. Given the direct interactions with both Pex15 and the D2 ATPase domains, the Pex6 N1 domain is poised to coordinate binding of cofactors and substrates with Pex1/Pex6 ATPase activity.

An AAA Motor-Driven Mechanical Switch in Rpn11 Controls Deubiquitination at the 26S Proteasome.

Poly-ubiquitin chains direct protein substrates to the 26S proteasome, where they are removed by the deubiquitinase Rpn11 during ATP-dependent substrate degradation. Rapid deubiquitination is required for efficient degradation but must be restricted to committed substrates that are engaged with the ATPase motor to prevent premature ubiquitin chain removal and substrate escape. Here we reveal the ubiquitin-bound structure of Rpn11 from S. cerevisiae and the mechanisms for mechanochemical coupling of substrate degradation and deubiquitination. Ubiquitin binding induces a conformational switch of Rpn11's Insert-1 loop from an inactive closed state to an active ß hairpin. This switch is rate-limiting for deubiquitination and strongly accelerated by mechanical substrate translocation into the AAA+ motor. Deubiquitination by Rpn11 and ubiquitin unfolding by the ATPases are in direct competition. The AAA+ motor-driven acceleration of Rpn11 is therefore important to ensure that poly-ubiquitin chains are removed only from committed substrates and fast enough to prevent their co-degradation.

Structures of asymmetric ClpX hexamers reveal nucleotide-dependent motions in a AAA+ protein-unfolding machine.

ClpX is a AAA+ machine that uses the energy of ATP binding and hydrolysis to unfold native proteins and translocate unfolded polypeptides into the ClpP peptidase. The crystal structures presented here reveal striking asymmetry in ring hexamers of nucleotide-free and nucleotide-bound ClpX. Asymmetry arises from large changes in rotation between the large and small AAA+ domains of individual subunits. These differences prevent nucleotide binding to two subunits, generate a staggered arrangement of ClpX subunits and pore loops around the hexameric ring, and provide a mechanism for coupling conformational changes caused by ATP binding or hydrolysis in one subunit to flexing motions of the entire ring. Our structures explain numerous solution studies of ClpX function, predict mechanisms for pore elasticity during translocation of irregular polypeptides, and suggest how repetitive conformational changes might be coupled to mechanical work during the ATPase cycle of ClpX and related molecular machines.

IR spectrum of SiH3OH2+SiH4: cationic OH⋯HSi dihydrogen bond versus charge-inverted SiH⋯Si hydrogen bond.

The low electronegativity of Si gives rise to a variety of nonconventional intermolecular interactions in clusters of silanes and their derivatives, which have not been well characterized yet. Herein, we characterize the structures of various isomers of bare and Ar-tagged SiH3OH2+SiH4 dimers composed of protonated silanol and silane by infrared photodissociation (IRPD) of mass-selected ions and dispersion-corrected density functional calculations (B3LYP-D3/aug-cc-pVTZ). The analysis of the IRPD spectra recorded in the OH stretch range reveals the competition between two types of nonconventional hydrogen bonds (H-bonds). The first one represents a OH⋯HSi ionic dihydrogen bond (DHB), in which SiH4 interacts with the H2O moiety of SiH3OH2+. The second one represents a charge-inverted SiH⋯Si ionic H-bond (CIHB), in which the SiH4 ligand interacts with the SiH3 moiety of SiH3OH2+. The latter may also be considered as a weak three-centre two-electron (3c-2e) bond. Although both types of H-bonds are computed to have comparable interaction strengths for SiH3OH2+SiH4 (D0 ≈ 35-40 kJ mol-1), DHB isomers dominate the population in the supersonic plasma expansion, while the abundance of CIHB isomers is roughly one order of magnitude lower, probably as a result of entropic factors.

Infrared spectra of SinH4n-1+ ions (n = 2-8): inorganic H-(Si-H)n-1 hydride wires of penta-coordinated Si in 3c-2e and charge-inverted hydrogen bonds.

SinHm+ cations are important constituents in silane plasmas and astrochemical environments. Protonated disilane (Si2H7+) was shown to have a symmetric three-centre two-electron (3c-2e) Si-H-Si bond that can also be considered as a strong ionic charge-inverted hydrogen bond with polarity Siδ+-Hδ--Siδ+. Herein, we extend our previous work to larger SinH4n-1+ cations, formally resulting from adding SiH4 molecules to a SiH3+ core. Infrared spectra of size-selected SinH4n-1+ ions (n = 2-8) produced in a cold SiH4/H2/He plasma expansion are analysed in the SiH stretch range by complementary dispersion-corrected density functional theory calculations (B3LYP-D3/aug-cc-pVTZ) to reveal their bonding characteristics and cluster growth. The ions with n = 2-4 form a linear inorganic H-(Si-H)n hydride wire with adjacent Si-H-Si 3c-2e bridges, whose strength decreases with n, as evident from their characteristic and strongly IR active SiH stretch fundamentals in the range 1850-2100 cm-1. These 3c-2e bonds result from the lowest-energy valence orbitals, and their high stability arises from their delocalization along the whole hydride wire. For SinH4n-1+ with n ≥ 5, the added SiH4 ligands form weak van der Waals bonds to the Si4H19+ chain. Significantly, because the SinH4n-1+ hydride wires are based on penta-coordinated Si atoms leading to supersaturated hydrosilane ions, analogous wires cannot be formed by isovalent carbon.

Exercise training to increase tumour natural killer-cell infiltration in men with localised prostate cancer: a randomised controlled trial.

OBJECTIVES: To explore the effects of preoperative high-intensity interval training (HIIT) compared to usual care on tumour natural killer (NK)-cell infiltration in men with localised prostate cancer (PCa), as NK-cell infiltration has been proposed as one of the key mechanisms whereby exercise can modulate human tumours. PATIENTS AND METHODS: A total of 30 patients with localised PCa undergoing radical prostatectomy (RP) were randomised (2:1) to either preoperative aerobic HIIT four-times weekly (EX; n = 20) or usual care (CON; n = 10) from time of inclusion until scheduled surgery. Tumour NK-cell infiltration was assessed by immunohistochemistry (CD56+ ) in diagnostic core needle biopsies and corresponding prostatic tissue from the RP. Changes in cardiorespiratory fitness, body composition, blood biochemistry, and health-related quality of life were also evaluated. RESULTS: The change in tumour NK-cell infiltration did not differ between the EX and CON groups (between-group difference: -0.09 cells/mm2 , 95% confidence interval [CI] -1.85 to 1.66; P = 0.913) in the intention-to-treat analysis. The total number of exercise sessions varied considerably from four to 30 sessions. The per-protocol analysis showed a significant increase in tumour NK-cell infiltration of 1.60 cells/mm2 (95% CI 0.59 to 2.62; P = 0.004) in the EX group. Further, the total number of training sessions was positively correlated with the change in NK-cell infiltration (r = 0.526, P = 0.021), peak oxygen uptake (r = 0.514, P = 0.035) and peak power output (r = 0.506, P = 0.038). CONCLUSION: Preoperative HIIT did not result in between-group differences in tumour NK-cell infiltration. Per-protocol and exploratory analyses demonstrate an enhanced NK-cell infiltration in PCa. Future studies are needed to test the capability of exercise to increase tumour immune cell infiltration.

Microhydrated clusters of a pharmaceutical drug: infrared spectra and structures of amantadineH+(H2O)n.

Solvation of pharmaceutical drugs has an important effect on their structure and function. Analysis of infrared photodissociation spectra of amantadineH+(H2O)n=1-4 clusters in the sensitive OH, NH, and CH stretch range by quantum chemical calculations (B3LYP-D3/cc-pVTZ) provides a first impression of the interaction of this pharmaceutically active cation with water at the molecular level. The size-dependent frequency shifts reveal detailed information about the acidity of the protons of the NH3+ group of N-protonated amantadineH+ (AmaH+) and the strength of the NH⋯O and OH⋯O hydrogen bonds (H-bonds) of the hydration network. The preferred cluster growth begins with sequential hydration of the NH3+ group by NH⋯O ionic H-bonds (n = 1-3), followed by the extension of the solvent network through OH⋯O H-bonds. However, smaller populations of cluster isomers with an H-bonded solvent network and free N-H bonds are already observed for n ≥ 2, indicating the subtle competition between noncooperative ion hydration and cooperative H-bonding. Interestingly, cyclic water ring structures are identified for n ≥ 3, each with two NH⋯O and two OH⋯O H-bonds. Despite the increasing destabilization of the N-H proton donor bonds upon gradual hydration, no proton transfer to the (H2O)n solvent cluster is observed up to n = 4. In addition to ammonium cluster ions, a small population of microhydrated iminium isomers is also detected, which is substantially lower for the hydrophilic H2O than for the hydrophobic Ar environment.

Microhydration of the adamantane cation: intracluster proton transfer to solvent in [Ad(H2O)n=1-5]+ for n ≥ 3.

Radical cations of diamondoids are important intermediates in their functionalization reactions in polar solvents. To explore the role of the solvent at the molecular level, we characterize herein microhydrated radical cation clusters of the parent molecule of the diamondoid family, adamantane (C10H16, Ad), by infrared photodissociation (IRPD) spectroscopy of mass-selected [Ad(H2O)n=1-5]+ clusters. IRPD spectra of the cation ground electronic state recorded in the CH/OH stretch and fingerprint ranges reveal the first steps of this fundamental H-substitution reaction at the molecular level. Analysis of size-dependent frequency shifts with dispersion-corrected density functional theory calculations (B3LYP-D3/cc-pVTZ) provides detailed information about the acidity of the proton of Ad+ as a function of the degree of hydration, the structure of the hydration shell, and the strengths of the CH⋯O and OH⋯O hydrogen bonds (H-bonds) of the hydration network. For n = 1, H2O strongly activates the acidic C-H bond of Ad+ by acting as a proton acceptor in a strong CH⋯O ionic H-bond with cation-dipole configuration. For n = 2, the proton is almost equally shared between the adamantyl radical (C10H15, Ady) and the (H2O)2 dimer in a strong C⋯H⋯O ionic H-bond. For n ≥ 3, the proton is completely transferred to the H-bonded hydration network. The threshold for this size-dependent intracluster proton transfer to solvent is consistent with the proton affinities of Ady and (H2O)n and confirmed by collision-induced dissociation experiments. Comparison with other related microhydrated cations reveals that the acidity of the CH proton of Ad+ is in the range of strongly acidic phenol+ but lower than for cationic linear alkanes such as pentane+. Significantly, the presented IRPD spectra of microhydrated Ad+ provide the first spectroscopic molecular-level insight of the chemical reactivity and reaction mechanism of the important class of transient diamondoid radical cations in aqueous solution.

A basidomycetous hydroxynaphthalene-prenylating enzyme exhibits promiscuity toward prenyl donors.

The fungal prenyltransferase ShPT from Stereum hirsutum was believed to prenylate 4-hydroxybenzyl alcohol and thereby be involved in the vibralactone biosynthesis. In this study, we demonstrate that hydroxynaphthalenes instead of benzyl alcohol or aldehyde were accepted by ShPT for regular C-prenylation in the presence of both dimethylallyl and geranyl diphosphate. Although the natural substrate of ShPT remains unknown, our results provide one additional prenyltransferase from basidiomycetes, which are less studied, in comparison to those from other sources. Furthermore, this study expands the chemical toolbox for regioselective production of prenylated naphthalene derivatives. KEY POINTS: •Basidiomycetous prenyltransferase •Biochemical characterization •A DMATS prenyltransferase prenylating hydroxynaphthalene derivatives.

The AAA+ ATPase Msp1 is a processive protein translocase with robust unfoldase activity.

Msp1 is a conserved eukaryotic AAA+ ATPase localized to the outer mitochondrial membrane, where it is thought to extract mislocalized tail-anchored proteins. Despite recent in vivo and in vitro studies supporting this function, a mechanistic understanding of how Msp1 extracts its substrates is still lacking. Msp1's ATPase activity depends on its hexameric state, and previous characterizations of the cytosolic AAA+ domain in vitro had proved challenging due to its monomeric nature in the absence of the transmembrane domain. Here, we used a hexamerization scaffold to study the substrate-processing mechanism of the soluble Msp1 motor, the functional homo-hexameric state of which was confirmed by negative-stain electron microscopy. We demonstrate that Msp1 is a robust bidirectional protein translocase that is able to unfold diverse substrates by processive threading through its central pore. This unfoldase activity is inhibited by Pex3, a membrane protein proposed to regulate Msp1 at the peroxisome.

Association between platelet lymphocyte ratio and neutrophil lymphocyte ratio and clinical outcomes following carotid endarterectomy.

Background: Approximately 30% of stroke cases result from carotid disease. Although several risk factors for complications after carotid endarterectomy have been identified, the existence of a biomarker that can estimate postoperative risk in these patients has not yet been proven. Objectives: This study aimed to investigate correlations between the platelet-lymphocyte ratio (PLR) and the neutrophil-lymphocyte ratio (NLR) and postoperative clinical outcomes in patients undergoing carotid endarterectomy. Methods: A retrospective study was conducted, including 374 patients who underwent carotid endarterectomy between 2002 and 2019 due to moderate to high extracranial internal carotid artery stenosis. Their platelet-lymphocyte ratio and neutrophil-lymphocyte ratios were obtained from the same blood samples. Results: There was a statistically significant correlation between the PLR and the occurrence of restenosis (p < 0.01) and acute myocardial infarction (AMI) after endarterectomy (p = 0.03). Additionally, there was a statistically significant correlation between the PLR and the combined outcomes stroke and/or AMI and/or death (p = 0.03) and stroke and/or AMI and/or death and/or restenosis (p < 0.01). However, there were no significant correlations between NLR and these outcomes (p = 0.05, p = 0.16). Conclusions: The platelet-lymphocyte ratio proved to be a useful test for predicting occurrence of strokes, acute myocardial infarctions, and deaths during the postoperative period after carotid endarterectomy. It was also associated with the risk of postoperative restenosis.

Contexto: Aproximadamente 30% dos casos de acidente vascular cerebral (AVC) resultam de doença carotídea. Embora vários fatores de risco para complicações pós-endarterectomia carotídea tenham sido identificados, ainda não foi comprovada a existência de um biomarcador que possa estimar o risco pós-operatório nesses pacientes. Objetivos: Correlacionar o índice plaqueta-linfócito (IPL) e o índice neutrófilo-linfócito (INL) com os desfechos clínicos pós-operatórios em pacientes submetidos a endarterectomia carotídea. Métodos: Estudo retrospectivo que incluiu 374 pacientes submetidos a endarterectomia carotídea, entre 2009 e 2019, por estenose extracraniana da artéria carótida interna. O IPL e o INL foram calculados, tendo sido obtidos das mesmas amostras de sangue. Resultados: Houve correlação estatisticamente significativa entre IPL e presença de reestenose (p<0,01) e infarto agudo do miocárdio (IAM) após endarterectomia (p=0,03). Os desfechos combinados AVC e/ou IAM e/ou óbito e AVC e/ou IAM e/ou óbito e/ou reestenose apresentaram, respectivamente, correlação estatisticamente significativa com o IPL (p=0,03; p<0,01) e não significativa com o INL (p=0,05; p=0,16). Conclusões: O IPL mostrou-se um teste útil, capaz de predizer os desfechos de AVC e/ou IAM e/ou óbito em pacientes no pós-operatório de endarterectomia carotídea, relacionando-se também com risco de reestenose pós-operatória.

Prescription rates for commonly used drugs before and after a prostate cancer diagnosis.

PURPOSE: To investigate differences in prescription rates of commonly used drugs among prostate cancer patients and cancer-free comparisons and between patients diagnosed with localized and non-localized disease. METHODS: We conducted a register-based study including all men aged 50-85 years diagnosed with prostate cancer in Denmark from 1998 to 2015 and an age-matched cancer-free comparison cohort. We calculated the number of new and total prescriptions from three years before to three years after the date of diagnosis of the case for selected drug classes divided by the number of person-months and stratified by stage at diagnosis. RESULTS: We included 54,286 prostate cancer patients and 249,645 matched comparisons. 30,712 patients were diagnosed with localized disease and 12,884 with non-localized disease. The rates of new prescriptions increased considerably among patients within the year before the diagnosis. Hereafter the rates varied between drug classes. For most drug classes, total prescription rates for patients and comparisons increased similarly in the study period. Total prescription rates varied between men with localized and non-localized disease for all drug classes apart from statins. CONCLUSION: Our findings indicate that a large proportion of prostate cancer cases are likely diagnosed during medical work-up for other reasons than prostate cancer. Increased rates occur within the last year before diagnosis and future studies on the interaction between drug use and prostate cancer should at least include a one year pre-diagnostic lag-time. Post-diagnostic prescription rates demonstrated an increased use of drugs most likely associated with the consequences of the disease.

A Nationwide Analysis of Risk of Prostate Cancer Diagnosis and Mortality following an Initial Negative Transrectal Ultrasound Biopsy with Long-Term Followup.

PURPOSE: Magnetic resonance imaging (MRI) targeted prostate biopsy has been shown to find many high-grade prostate cancers in men with concurrent negative transrectal ultrasound (TRUS) systematic biopsy. The oncologic risk of such tumors can be explored by looking at long-term outcomes of men with negative TRUS biopsy followed without MRI. The aim was to analyze the mortality after initial and second negative TRUS biopsy. MATERIALS AND METHODS: All men who underwent initial TRUS biopsies between January 1, 1995 and December 31, 2016 in Denmark were included. A total of 37,214 men had a negative initial TRUS biopsy and 6,389 underwent a re-biopsy. Risk of cause-specific mortality was analyzed with competing risks. Diagnosis of Gleason score ≥7 prostate cancer following negative biopsies was analyzed with multivariable logistic regression including time to re-biopsy, prostate specific antigen (PSA), age and digital rectal examination. RESULTS: The 15-year prostate cancer-specific mortality was 1.9% (95% CI: 1.7-2.1). Prostate cancer-specific mortality was 1.3% (95% CI: 0.9-1.6) and 4.6% (95% CI: 3.4-5.8) for men with PSA <10 and >20 ng/ml, respectively. Of the TRUS re-biopsies 12% were Gleason score ≥7 and risk of Gleason score ≥7 increased with longer time to re-biopsy (p <0.001). Mortality after re-biopsy was similar to after initial biopsy. CONCLUSIONS: Men with negative TRUS biopsies have a very low prostate cancer-specific mortality, especially with PSA <10 ng/ml. This raises serious questions about the routine use of MRI targeting for initial prostate biopsy and suggests that MRI targeting should only be recommended for men with PSA >10 ng/ml after negative biopsy.