Distal Radial Artery Access for Coronary and Peripheral Procedures: A Multicenter Experience.

INTRODUCTION: Distal radial access (dRA) has recently gained global popularity as an alternative access route for vascular procedures. Among the benefits of dRA are the low risk of entry site bleeding complications, the low rate of radial artery occlusion, and improved patient and operator comfort. The aim of this large multicenter registry was to demonstrate the feasibility and safety of dRA in a wide variety of routine procedures in the catheterization laboratory, ranging from coronary angiography and percutaneous coronary intervention to peripheral procedures. METHODS: The study comprised 1240 patients who underwent coronary angiography, PCI or noncoronary procedures through dRA in two Hungarian centers from January 2019 to April 2021. Baseline patient characteristics, number and duration of arterial punctures, procedural success rate, crossover rate, postoperative compression time, complications, hospitalization duration, and different learning curves were analyzed. RESULTS: The average patient age was 66.4 years, with 66.8% of patients being male. The majority of patients (74.04%) underwent a coronary procedure, whereas 25.96% were involved in noncoronary interventions. dRA was successfully punctured in 97% of all patients, in all cases with ultrasound guidance. Access site crossover was performed in 2.58% of the patients, mainly via the contralateral dRA. After experiencing 150 cases, the dRA success rate plateaued at >96%. Our dedicated dRA step-by step protocol resulted in high open radial artery (RA) rates: distal and proximal RA pulses were palpable in 99.68% of all patients at hospital discharge. The rate of minor vascular complications was low (1.5%). A threshold of 50 cases was sufficient for already skilled radial operators to establish a reliable procedural method of dRA access. CONCLUSION: The implementation of distal radial artery access in the everyday routine of a catheterization laboratory for coronary and noncoronary interventions is feasible and safe with an acceptable learning curve.

Finding the optimal access for proximal upper limb artery (PULA) interventions: Lessons learned from the PULA multicenter registry.

OBJECTIVE: The multicenter proximal upper limb artery (PULA) Registry was created to study the optimal puncture sites for the interventions involving the subclavian, axillary, and innominate arteries. BACKGROUND: Little is known about the optimal vascular access for PULA interventions, despite the well-known technical complexity of these procedures. METHODS: We performed the retrospective analysis of consecutive patients treated for symptomatic steno-occlusive disease of the proximal upper limb arteries between January 2015 and December 2019 in three high-volume centers. Acute thrombotic occlusions were excluded from the study. RESULTS: Two hundred and seventy-two patients were treated for significant stenosis and 108 for total occlusion. The baseline patient's characteristics were similar, except for the higher median age of the stenotic patients: 68.5 years (31.1; 90.0) versus 64 years (38.0; 86.0) p = 0.0015. Successful revascularization rate was higher in the stenotic group 93.75% (255/272) versus 86.11% (93/108) p = 0.0230, while the procedure length 27 min (8; 133) versus 46 min (7; 140) p = 0.0001 and fluoroscopy times 439 s (92; 2993) versus 864 s (86; 4176) p = 0.0001 were higher in the occlusion group. The main adverse event rate was similarly low. Dual access was used more often to treat occlusions (60.19% (65/108) vs. 11.40% (31/272) p = 0.0001) without significantly increasing the complication rate. The safest access was ultrasound-guided distal radial artery puncture, significantly better than conventional radial access with 0% (0/31) versus 13.6% (18/131) p = 0.0253 complication. CONCLUSIONS: The percutaneous revascularization of proximal upper limb arteries is a safe and effective. Dual access can be applied to increase treatment efficacy, without significantly compromising safety.

Short- and long-term results with a percutaneous treatment in critical hand ischaemia.

BACKGROUND: The aim of this prospective registry was to determine the feasibility, safety, and outcomes of percutaneous transluminal angioplasty and thrombolysis in the treatment of critical hand ischemia (CHI). METHODS: One-hundred one patients (aged 60.6 ± 15.3 years) were treated for CHI between 2012 and 2016 in three cardiovascular centers. Anatomically, the upper arm was divided into three segments (I-subclavian, II-brachial, and III-forearm). We examined the rates of technical and clinical success, major adverse events (MAEs), and vascular complications at 1 year and at long-term follow-up. RESULTS: Nineteen patients (18.8%) were treated for acute CHI, and 82 (81.2%) for chronic CHI. Median follow-up was 36.9 (19.6-68.3) months. Clinical symptoms were isolated rest pain in 91 patients (90.1%) and digital ulcer or gangrene in 10 patients (9.9%). The technical and clinical success rate of intervention was 96.0% (97/101) and 84.2% (85/101) at 1 year. Angioplasty was performed in Segments I, II, and III in 28 (27.7%), in 29 (28.7%), and 44 (43.5%) patients. Stent implantation was necessary in 47 patients (46.8%). Vascular access site complications were found in 2.1% of the sample. After 1 year, MAEs occurred in 27 patients (26.9%), and the target lesion revascularization rate was 11.9%. In two patients (1.9%), thoracic sympatectomy was necessary, and two patients (1.9%) underwent minor finger amputations. CONCLUSIONS: Angioplasty of hand vessels for CHI is a feasible and safe procedure with acceptable rates of technical success and hand healing. MAEs are frequent because the rate of severe comorbidities is high.

Adapted ATPase domain communication overcomes the cytotoxicity of p97 inhibitors.

The AAA+ ATPase p97 regulates ubiquitin-dependent protein homeostasis and has been pursued as a cancer drug target. The ATP-competitive inhibitor CB-5083 and allosteric inhibitor NMS-873 are the most advanced p97 inhibitors described to date. Previous studies have reported that their cytotoxicity can be readily overcome and involves single p97 mutations in the linker between the D1 and D2 ATPase domains and within D2. We report here that the proline 472 to leucine (P472L) mutation, in the D1-D2 linker and identified in CB-5083-resistant cells, desensitizes p97 to both inhibitor classes. This mutation does not disrupt the distinct D2-binding sites of the inhibitors. Instead, P472L changes ATPase domain communication within the p97 hexamer. P472L enhances cooperative D2 ATP binding and hydrolysis. This mechanism alters the function of the D1-D2 linker in the control of D2 activity involving the ATP-bound state of D1. Although increased D2 activity is sufficient to desensitize the P472L mutant to NMS-873, the mutant's desensitization to CB-5083 also requires D1 ATPase domain function. Our study highlights the remarkable adaptability of p97 ATPase domain communication that enables escape from mechanistically distinct classes of cytotoxic p97 inhibitors.

p97 Composition Changes Caused by Allosteric Inhibition Are Suppressed by an On-Target Mechanism that Increases the Enzyme's ATPase Activity.

The AAA ATPase p97/VCP regulates protein homeostasis using a diverse repertoire of cofactors to fulfill its biological functions. Here we use the allosteric p97 inhibitor NMS-873 to analyze its effects on enzyme composition and the ability of cells to adapt to its cytotoxicity. We found that p97 inhibition changes steady state cofactor-p97 composition, leading to the enrichment of a subset of its cofactors and polyubiquitin bound to p97. We isolated cells specifically insensitive to NMS-873 and identified a new mutation (A530T) in p97. A530T is sufficient to overcome the cytotoxicity of NMS-873 and alleviates p97 composition changes caused by the molecule but not other p97 inhibitors. This mutation does not affect NMS-873 binding but increases p97 catalytic efficiency through altered ATP and ADP binding. Collectively, these findings identify cofactor-p97 interactions sensitive to p97 inhibition and reveal a new on-target mechanism to suppress the cytotoxicity of NMS-873.

Re-evaluating the kinetics of ATP hydrolysis during initiation of DNA sliding by Type III restriction enzymes.

DNA cleavage by the Type III restriction enzymes requires long-range protein communication between recognition sites facilitated by thermally-driven 1D diffusion. This 'DNA sliding' is initiated by hydrolysis of multiple ATPs catalysed by a helicase-like domain. Two distinct ATPase phases were observed using short oligoduplex substrates; the rapid consumption of ∼10 ATPs coupled to a protein conformation switch followed by a slower phase, the duration of which was dictated by the rate of dissociation from the recognition site. Here, we show that the second ATPase phase is both variable and only observable when DNA ends are proximal to the recognition site. On DNA with sites more distant from the ends, a single ATPase phase coupled to the conformation switch was observed and subsequent site dissociation required little or no further ATP hydrolysis. The overall DNA dissociation kinetics (encompassing site release, DNA sliding and escape via a DNA end) were not influenced by the second phase. Although the data simplifies the ATP hydrolysis scheme for Type III restriction enzymes, questions remain as to why multiple ATPs are hydrolysed to prepare for DNA sliding.

Mutations in UBA3 confer resistance to the NEDD8-activating enzyme inhibitor MLN4924 in human leukemic cells.

The NEDD8-activating enzyme (NAE) initiates neddylation, the cascade of post-translational NEDD8 conjugation onto target proteins. MLN4924, a selective NAE inhibitor, has displayed preclinical anti-tumor activity in vitro and in vivo, and promising clinical activity has been reported in patients with refractory hematologic malignancies. Here, we sought to understand the mechanisms of resistance to MLN4924. K562 and U937 leukemia cells were exposed over a 6 month period to MLN4924 and populations of resistant cells (R-K562(MLN), R-U937(MLN)) were selected. R-K562(MLN) and R-U937(MLN) cells contain I310N and Y352H mutations in the NAE catalytic subunit UBA3, respectively. Biochemical analyses indicate that these mutations increase the enzyme's affinity for ATP while decreasing its affinity for NEDD8. These mutations effectively contribute to decreased MLN4924 potency in vitro while providing for sufficient NAE function for leukemia cell survival. Finally, R-K562(MLN) cells showed cross-resistance to other NAE-selective inhibitors, but remained sensitive to a pan-E1 (activating enzyme) inhibitor. Thus, our work provides insight into mechanisms of MLN4924 resistance to facilitate the development of more effective second-generation NAE inhibitors.

Type III restriction endonucleases are heterotrimeric: comprising one helicase-nuclease subunit and a dimeric methyltransferase that binds only one specific DNA.

Fundamental aspects of the biochemistry of Type III restriction endonucleases remain unresolved despite being characterized by numerous research groups in the past decades. One such feature is the subunit stoichiometry of these hetero-oligomeric enzyme complexes, which has important implications for the reaction mechanism. In this study, we present a series of results obtained by native mass spectrometry and size exclusion chromatography with multi-angle light scattering consistent with a 1:2 ratio of Res to Mod subunits in the EcoP15I, EcoPI and PstII complexes as the main holoenzyme species and a 1:1 stoichiometry of specific DNA (sDNA) binding by EcoP15I and EcoPI. Our data are also consistent with a model where ATP hydrolysis activated by recognition site binding leads to release of the enzyme from the site, dissociation from the substrate via a free DNA end and cleavage of the DNA. These results are discussed critically in the light of the published literature, aiming to resolve controversies and discuss consequences in terms of the reaction mechanism.

N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells.

N(6)-methyladenosine (m(6)A) has been identified as the most abundant internal modification of messenger RNA in eukaryotes. m(6)A modification is involved in cell fate determination in yeast and embryo development in plants. Its mammalian function remains unknown but thousands of mammalian mRNAs and long non-coding RNAs (lncRNAs) show m(6)A modification and m(6)A demethylases are required for mammalian energy homeostasis and fertility. We identify two proteins, the putative m(6)A MTase, methyltransferase-like 3 (Mettl3; ref. ), and a related but uncharacterized protein Mettl14, that function synergistically to control m(6)A formation in mammalian cells. Knockdown of Mettl3 and Mettl14 in mouse embryonic stem cells (mESCs) led to similar phenotypes, characterized by lack of m(6)A RNA methylation and lost self-renewal capability. A large number of transcripts, including many encoding developmental regulators, exhibit m(6)A methylation inversely correlated with mRNA stability and gene expression. The human antigen R (HuR) and microRNA pathways were linked to these effects. This gene regulatory mechanism operating in mESCs through m(6)A methylation is required to keep mESCs at their ground state and may be relevant to thousands of mRNAs and lncRNAs in various cell types.

Lys11- and Lys48-linked ubiquitin chains interact with p97 during endoplasmic-reticulum-associated degradation.

The ATPase associated with various cellular activities p97 has a critical function in the cytoplasmic degradation of proteins misfolded in the ER (endoplasmic reticulum) through a mechanism known as ERAD (ER-associated degradation). During this process, p97 binds polyubiquitinated ERAD substrates and couples ATP hydrolysis to their dislocation from the ER as a prerequisite to destruction by the proteasome. The ubiquitin signals important for this process are not fully understood. In the present paper we report that p97 interacts with Lys11- and Lys48-linked ubiquitin polymers, but not those containing Lys63 linkages. Disruption of p97 through siRNA-mediated depletion, dominant-negative overexpression or chemical inhibition results in the accumulation of Lys11 and Lys48 ubiquitin chains predominantly at the ER membrane, and is associated with ER stress induction. We show that a catalytically inactive deubiquitinating enzyme and p97 cofactor YOD1 enhances the accumulation of Lys11- and Lys48-linked polyubiquitin in the cytoplasm, at the ER membrane and bound to p97. In addition to general effects on p97-associated ubiquitin polymers, the ERAD substrate CD3δ is modified with both Lys11 and Lys48 ubiquitin chains prior to p97-dependent dislocation. Collectively, the results of the present study are consistent with a major role for p97 in the recognition of Lys11 and Lys48 polyubiquitinated proteins before their degradation by the proteasome.

The cyclomodulin cycle inhibiting factor (CIF) alters cullin neddylation dynamics.

The bacterial effector protein cycle inhibiting factor (CIF) converts glutamine 40 of NEDD8 to glutamate (Q40E), causing cytopathic effects and inhibiting cell proliferation. Although these have been attributed to blocking the functions of cullin-RING ubiquitin ligases, how CIF modulates NEDD8-dependent signaling is unclear. Here we use conditional NEDD8-dependent yeast to explore the effects of CIF on cullin neddylation. Although CIF causes cullin deneddylation and the generation of free NEDD8 Q40E, inhibiting the COP9 signalosome (CSN) allows Q40E to form only on NEDD8 attached to cullins. In the presence of the CSN, NEDD8 Q40E is removed from cullins more rapidly than NEDD8, leading to a decrease in steady-state cullin neddylation. As NEDD8 Q40E is competent for cullin conjugation in the absence of functional CSN and with overexpression of the NEDD8 ligase Dcn1, our data are consistent with NEDD8 deamidation causing enhanced deneddylation of cullins by the CSN. This leads to a dramatic change in the extent of activated cullin-RING ubiquitin ligases.

The helicase-like domains of type III restriction enzymes trigger long-range diffusion along DNA.

Helicases are ubiquitous adenosine triphosphatases (ATPases) with widespread roles in genome metabolism. Here, we report a previously undescribed functionality for ATPases with helicase-like domains; namely, that ATP hydrolysis can trigger ATP-independent long-range protein diffusion on DNA in one dimension (1D). Specifically, using single-molecule fluorescence microscopy we show that the Type III restriction enzyme EcoP15I uses its ATPase to switch into a distinct structural state that diffuses on DNA over long distances and long times. The switching occurs only upon binding to the target site and requires hydrolysis of ~30 ATPs. We define the mechanism for these enzymes and show how ATPase activity is involved in DNA target site verification and 1D signaling, roles that are common in DNA metabolism: for example, in nucleotide excision and mismatch repair.

CAND1 controls in vivo dynamics of the cullin 1-RING ubiquitin ligase repertoire.

The combinatorial architecture of cullin 1-RING ubiquitin ligases, in which multiple F-box containing substrate receptors compete for access to CUL1, poses special challenges to assembling cullin 1-RING ubiquitin ligase complexes through high affinity protein interactions while maintaining the flexibility to dynamically sample the entire F-box containing substrate receptor repertoire. Here, using highly quantitative mass spectrometry, we demonstrate that this problem is addressed by CAND1, a factor that controls the dynamics of the global cullin 1-RING ubiquitin ligase network by promoting the assembly of newly synthesized F-box containing substrate receptors with CUL1-RBX1 core complexes. Our studies of in vivo cullin 1-RING ubiquitin ligase dynamics and in vitro biochemical findings showing that CAND1 can displace F-box containing substrate receptors from Cul1p suggest that CAND1 functions in a cycle that serves to exchange F-box containing substrate receptors on CUL1 cores. We propose that this cycle assures comprehensive sampling of the entire F-box containing substrate receptor repertoire in order to maintain the cullin 1-RING ubiquitin ligase landscape, a function that we show to be critical for substrate degradation and normal physiology.

A gatekeeper residue for NEDD8-activating enzyme inhibition by MLN4924.

Inhibition of NEDD8-activating enzyme (NAE) has emerged as a highly promising approach to treat cancer through the adenosine sulfamate analog MLN4924. Here, we show that selective pressure results in HCT116 colorectal carcinoma cells with decreased MLN4924 sensitivity and identify a single-nucleotide transition that changes alanine 171 to threonine (A171T) of the NAE subunit UBA3. This reduces the enzyme's affinity for MLN4924 and ATP while increasing NEDD8 activation at physiological ATP concentrations. Expression of UBA3 A171T is sufficient to decrease MLN4924 sensitivity of naive HCT116 cells, indicating that it is a dominant suppressor of MLN4924-mediated cell death. Our data suggest that the on-target potency of MLN4924 selects for a point mutation in NAE that overcomes the molecule's inhibitory effects, allowing cancer cell survival.

Dissociation from DNA of Type III Restriction-Modification enzymes during helicase-dependent motion and following endonuclease activity.

DNA cleavage by the Type III Restriction-Modification (RM) enzymes requires the binding of a pair of RM enzymes at two distant, inversely orientated recognition sequences followed by helicase-catalysed ATP hydrolysis and long-range communication. Here we addressed the dissociation from DNA of these enzymes at two stages: during long-range communication and following DNA cleavage. First, we demonstrated that a communicating species can be trapped in a DNA domain without a recognition site, with a non-specific DNA association lifetime of ∼ 200 s. If free DNA ends were present the lifetime became too short to measure, confirming that ends accelerate dissociation. Secondly, we observed that Type III RM enzymes can dissociate upon DNA cleavage and go on to cleave further DNA molecules (they can 'turnover', albeit inefficiently). The relationship between the observed cleavage rate and enzyme concentration indicated independent binding of each site and a requirement for simultaneous interaction of at least two enzymes per DNA to achieve cleavage. In light of various mechanisms for helicase-driven motion on DNA, we suggest these results are most consistent with a thermally driven random 1D search model (i.e. 'DNA sliding').

DNA cleavage site selection by Type III restriction enzymes provides evidence for head-on protein collisions following 1D bidirectional motion.

DNA cleavage by the Type III Restriction-Modification enzymes requires communication in 1D between two distant indirectly-repeated recognitions sites, yet results in non-specific dsDNA cleavage close to only one of the two sites. To test a recently proposed ATP-triggered DNA sliding model, we addressed why one site is selected over another during cleavage. We examined the relative cleavage of a pair of identical sites on DNA substrates with different distances to a free or protein blocked end, and on a DNA substrate using different relative concentrations of protein. Under these conditions a bias can be induced in the cleavage of one site over the other. Monte-Carlo simulations based on the sliding model reproduce the experimentally observed behaviour. This suggests that cleavage site selection simply reflects the dynamics of the preceding stochastic enzyme events that are consistent with bidirectional motion in 1D and DNA cleavage following head-on protein collision.

Bifunctional apoptosis regulator (BAR), an endoplasmic reticulum (ER)-associated E3 ubiquitin ligase, modulates BI-1 protein stability and function in ER Stress.

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates inositol-requiring protein-1 (IRE1), among other ER-associated signaling proteins of the unfolded protein response (UPR) in mammalian cells. IRE1 signaling becomes attenuated under prolonged ER stress. The mechanisms by which this occurs are not well understood. An ER resident protein, Bax inhibitor-1 (BI-1), interacts with IRE1 and directly inhibits IRE1 activity. However, little is known about regulation of the BI-1 protein. We show here that bifunctional apoptosis regulator (BAR) functions as an ER-associated RING-type E3 ligase, interacts with BI-1, and promotes proteasomal degradation of BI-1. Overexpression of BAR reduced BI-1 protein levels in a RING-dependent manner. Conversely, knockdown of endogenous BAR increased BI-1 protein levels and enhanced inhibition of IRE1 signaling during ER stress. We also found that the levels of endogenous BAR were reduced under prolonged ER stress. Our findings suggest that post-translational regulation of the BI-1 protein by E3 ligase BAR contributes to the dynamic control of IRE1 signaling during ER stress.

Type III restriction enzymes cleave DNA by long-range interaction between sites in both head-to-head and tail-to-tail inverted repeat.

Cleavage of viral DNA by the bacterial Type III Restriction-Modification enzymes requires the ATP-dependent long-range communication between a distant pair of DNA recognition sequences. The classical view is that Type III endonuclease activity is only activated by a pair of asymmetric sites in a specific head-to-head inverted repeat. Based on this assumption and due to the presence of helicase domains in Type III enzymes, various motor-driven DNA translocation models for communication have been suggested. Using both single-molecule and ensemble assays we demonstrate that Type III enzymes can also cleave DNA with sites in tail-to-tail repeat with high efficiency. The ability to distinguish both inverted repeat substrates from direct repeat substrates in a manner independent of DNA topology or accessory proteins can only be reconciled with an alternative sliding mode of communication.

Engineering novel complement activity into a pulmonary surfactant protein.

Complement neutralizes invading pathogens, stimulates inflammatory and adaptive immune responses, and targets non- or altered-self structures for clearance. In the classical and lectin activation pathways, it is initiated when complexes composed of separate recognition and activation subcomponents bind to a pathogen surface. Despite its apparent complexity, recognition-mediated activation has evolved independently in three separate protein families, C1q, mannose-binding lectins (MBLs), and serum ficolins. Although unrelated, all have bouquet-like architectures and associate with complement-specific serine proteases: MBLs and ficolins with MBL-associated serine protease-2 (MASP-2) and C1q with C1r and C1s. To examine the structural requirements for complement activation, we have created a number of novel recombinant rat MBLs in which the position and orientation of the MASP-binding sites have been changed. We have also engineered MASP binding into a pulmonary surfactant protein (SP-A), which has the same domain structure and architecture as MBL but lacks any intrinsic complement activity. The data reveal that complement activity is remarkably tolerant to changes in the size and orientation of the collagenous stalks of MBL, implying considerable rotational and conformational flexibility in unbound MBL. Furthermore, novel complement activity is introduced concurrently with MASP binding in SP-A but is uncontrolled and occurs even in the absence of a carbohydrate target. Thus, the active rather than the zymogen state is default in lectin.MASP complexes and must be inhibited through additional regions in circulating MBLs until triggered by pathogen recognition.