BBB opening with focused ultrasound in nonhuman primates and Parkinson's disease patients: Targeted AAV vector delivery and PET imaging.

Intracerebral vector delivery in nonhuman primates has been a major challenge. We report successful blood-brain barrier opening and focal delivery of adeno-associated virus serotype 9 vectors into brain regions involved in Parkinson's disease using low-intensity focus ultrasound in adult macaque monkeys. Openings were well tolerated with generally no associated abnormal magnetic resonance imaging signals. Neuronal green fluorescent protein expression was observed specifically in regions with confirmed blood-brain barrier opening. Similar blood-brain barrier openings were safely demonstrated in three patients with Parkinson's disease. In these patients and in one monkey, blood-brain barrier opening was followed by 18F-Choline uptake in the putamen and midbrain regions based on positron emission tomography. This indicates focal and cellular binding of molecules that otherwise would not enter the brain parenchyma. The less-invasive nature of this methodology could facilitate focal viral vector delivery for gene therapy and might allow early and repeated interventions to treat neurodegenerative disorders.

Combined Stimulation of the Substantia Nigra and the Subthalamic Nucleus for the Treatment of Refractory Gait Disturbances in Parkinson's Disease: A Preliminary Study.

Deep brain stimulation of the subthalamic nucleus is efficient for the treatment of motor symptoms (i.e., tremors) in patients with Parkinson's disease. Gait disorders usually appear during advanced stages of idiopathic Parkinson's disease in up to 80% of patients and have an important impact on their quality of life. The effects of deep brain stimulation of the subthalamic nucleus on gait and balance are still controversial. For this reason, alternative targets have been considered, such as stimulation of the pedunculopontine nucleus and the pars reticulata of substantia nigra, involved in the integration of the functional connections for gait. Due to the proximity of the subthalamic nucleus to the substantia nigra, their combined stimulation is feasible and may lead to better outcomes, improving axial symptoms. Our objective was to prospectively compare simultaneous stimulation of both structures versus conventional subthalamic stimulation in improving gait disorders. In ten patients with advanced Parkinson's disease, deep brain stimulation leads (eight linear contacts) were implanted, and gait analysis was performed 6 months after surgery in off-stimulation and after 4 weeks of dual or single subthalamic stimulation. An improvement in gait parameters was confirmed with both stimulation conditions, with better results with combined substantia nigra and subthalamic stimulation compared with conventional subthalamic stimulation. Further studies are needed to determine if this effect remains after long-term dual-target stimulation.

Lesion 3D modeling in transcranial MR-guided focused ultrasound thalamotomy.

Transcranial magnetic resonance-guided focused ultrasound (tMRgFUS) allows to perform incisionless thermoablation of deep brain structures. This feature makes it a very useful tool for the treatment of multiple neurological and psychiatric disorders. Currently, feedback of the thermoablation process is based on peak temperature readings assessed on real-time two-dimensional MRI thermometry. However, an accurate methodology relating thermal dosimetry with three-dimensional topography and temporal evolution of the lesion is still to be defined, thus hurdling the establishment of well-defined, evidence-based criteria to perform safe and effective treatments. In here we propose threshold-based thermoablation models to predict the volumetric topography of the lesion (whole lesion and necrotic core) in the short-to-mid-term based on thermal dosimetry estimated from intra-treatment MRI thermometry. To define and validate our models we retrospectively analyzed the data of sixty-three tMRgFUS thalamotomies for treating tremor. We used intra-treatment MRI thermometry to estimate whole-treatment three-dimensional thermal dose maps, defined either as peak temperature reached (Tmax) or thermal isoeffective dose (TID). Those maps were thresholded to find the dosimetric level that maximize the agreement (Sorensen-Dice coefficient - SDc) with the boundaries of the whole lesion and its core, assessed on T2w images 1-day (post-24h) and 3-months (post-3M) after treatment. Best predictions were achieved for the whole lesion at post-24h (SDc = 0.71), with Tmax /TID over 50.0 °C/90.5 CEM43. The core at post-24h and whole lesion at post-3M lesions reported a similar behavior in terms of shape accuracy (SDc ~0.35), and thermal dose thresholds ~55 °C/4100.0 CEM43. Finally, the optimal levels for post-3M core lesions were 55.5 °C/5800.0 CEM43 (SDc = 0.21). These thermoablation models could contribute to the real-time decision-making process and improve the outcome of tMRgFUS interventions both in terms of safety and efficacy.

Blood-brain barrier opening with focused ultrasound in Parkinson's disease dementia.

MR-guided focused ultrasound (MRgFUS), in combination with intravenous microbubble administration, has been applied for focal temporary BBB opening in patients with neurodegenerative disorders and brain tumors. MRgFUS could become a therapeutic tool for drug delivery of putative neurorestorative therapies. Treatment for Parkinson's disease with dementia (PDD) is an important unmet need. We initiated a prospective, single-arm, non-randomized, proof-of-concept, safety and feasibility phase I clinical trial (NCT03608553), which is still in progress. The primary outcomes of the study were to demonstrate the safety, feasibility and reversibility of BBB disruption in PDD, targeting the right parieto-occipito-temporal cortex where cortical pathology is foremost in this clinical state. Changes in ß-amyloid burden, brain metabolism after treatments and neuropsychological assessments, were analyzed as exploratory measurements. Five patients were recruited from October 2018 until May 2019, and received two treatment sessions separated by 2-3 weeks. The results are set out in a descriptive manner. Overall, this procedure was feasible and reversible with no serious clinical or radiological side effects. We report BBB opening in the parieto-occipito-temporal junction in 8/10 treatments in 5 patients as demonstrated by gadolinium enhancement. In all cases the procedures were uneventful and no side effects were encountered associated with BBB opening. From pre- to post-treatment, mild cognitive improvement was observed, and no major changes were detected in amyloid or fluorodeoxyglucose PET. MRgFUS-BBB opening in PDD is thus safe, reversible, and can be performed repeatedly. This study provides encouragement for the concept of BBB opening for drug delivery to treat dementia in PD and other neurodegenerative disorders.

Randomized Trial of Focused Ultrasound Subthalamotomy for Parkinson's Disease.

BACKGROUND: The subthalamic nucleus is the preferred neurosurgical target for deep-brain stimulation to treat cardinal motor features of Parkinson's disease. Focused ultrasound is an imaging-guided method for creating therapeutic lesions in deep-brain structures, including the subthalamic nucleus. METHODS: We randomly assigned, in a 2:1 ratio, patients with markedly asymmetric Parkinson's disease who had motor signs not fully controlled by medication or who were ineligible for deep-brain stimulation surgery to undergo focused ultrasound subthalamotomy on the side opposite their main motor signs or a sham procedure. The primary efficacy outcome was the between-group difference in the change from baseline to 4 months in the Movement Disorder Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS) motor score (i.e., part III) for the more affected body side (range, 0 to 44, with higher scores indicating worse parkinsonism) in the off-medication state. The primary safety outcome (procedure-related complications) was assessed at 4 months. RESULTS: Among 40 enrolled patients, 27 were assigned to focused ultrasound subthalamotomy (active treatment) and 13 to the sham procedure (control). The mean MDS-UPDRS III score for the more affected side decreased from 19.9 at baseline to 9.9 at 4 months in the active-treatment group (least-squares mean difference, 9.8 points; 95% confidence interval [CI], 8.6 to 11.1) and from 18.7 to 17.1 in the control group (least-squares mean difference, 1.7 points; 95% CI, 0.0 to 3.5); the between-group difference was 8.1 points (95% CI, 6.0 to 10.3; P<0.001). Adverse events in the active-treatment group were dyskinesia in the off-medication state in 6 patients and in the on-medication state in 6, which persisted in 3 and 1, respectively, at 4 months; weakness on the treated side in 5 patients, which persisted in 2 at 4 months; speech disturbance in 15 patients, which persisted in 3 at 4 months; facial weakness in 3 patients, which persisted in 1 at 4 months; and gait disturbance in 13 patients, which persisted in 2 at 4 months. In 6 patients in the active-treatment group, some of these deficits were present at 12 months. CONCLUSIONS: Focused ultrasound subthalamotomy in one hemisphere improved motor features of Parkinson's disease in selected patients with asymmetric signs. Adverse events included speech and gait disturbances, weakness on the treated side, and dyskinesia. (Funded by Insightec and others; ClinicalTrials.gov number, NCT03454425.).

Neuromuscular involvement in COVID-19 critically ill patients.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has a high incidence of intensive care admittance due to the severe acute respiratory syndrome (SARS). Intensive care unit (ICU)-acquired weakness (ICUAW) is a common complication of ICU patients consisting of symmetric and generalised weakness. The aim of this study was to determine the presence of myopathy, neuropathy or both in ICU patients affected by COVID-19 and whether ICUAW associated with COVID-19 differs from other aetiologies. METHODS: Twelve SARS CoV-2 positive patients referred with the suspicion of critical illness myopathy (CIM) or polyneuropathy (CIP) were included between March and May 2020. Nerve conduction and concentric needle electromyography were performed in all patients while admitted to the hospital. Muscle biopsies were obtained in three patients. RESULTS: Four patients presented signs of a sensory-motor axonal polyneuropathy and seven patients showed signs of myopathy. One muscle biopsy showed scattered necrotic and regenerative fibres without inflammatory signs. The other two biopsies showed non-specific myopathic findings. CONCLUSIONS: We have not found any distinctive features in the studies of the ICU patients affected by SARS-CoV-2 infection. SIGNIFICANCE: Further studies are needed to determine whether COVID-19-related CIM/CIP has different features from other aetiologies. Neurophysiological studies are essential in the diagnosis of these patients.

Transcranial Magnetic Resonance-Guided Focused Ultrasound Thalamotomy in Essential Tremor: A Comprehensive Lesion Characterization.

BACKGROUND: Transcranial magnetic resonance-guided focused ultrasound (tcMRgFUS) thalamotomy is a novel and effective treatment for controlling tremor in essential tremor patients. OBJECTIVE: To provide a comprehensive characterization of the radiological, topographical, and volumetric aspects of the tcMRgFUS thalamic lesion, and to quantify how they relate to the clinical outcomes. METHODS: In this study, clinical and radiological data from forty patients with medically-refractory essential tremor treated with unilateral tcMRgFUS thalamotomy were retrospectively analyzed. Treatment efficacy was assessed with Clinical Rating Scale for Tremor (CRST). Lesions were manually segmented on T1, T2, and susceptibility-weighted images, and 3-dimensional topographical analysis was then carried out. Statistical comparisons were performed using nonparametric statistics. RESULTS: The greatest clinical improvement was correlated with a more inferior and posterior lesion, a bigger lesion volume, and percentage of the ventral intermediate nucleus covered by the lesion; whereas, the largest lesions accounted for the occurrence of gait imbalance. Furthermore, the volume of the lesion was significantly predicted by the number of sonications surpassing 52°C. CONCLUSION: Here we provide a comprehensive characterization of the thalamic tcMRgFUS lesion including radiological and topographical analysis. Our results indicate that the location and volume of the lesion were significantly associated with the clinical outcome and that mid-temperatures may be responsible for the lesion size. This could serve ultimately to improve targeting and judgment and to optimize clinical outcome of tcMRgFUS thalamotomy.

The role of high-intensity focused ultrasound as a symptomatic treatment for Parkinson's disease.

MR-guided focused ultrasound is a novel, minimally invasive surgical procedure for symptomatic treatment of PD. With this technology, the ventral intermediate nucleus, STN, and internal globus pallidus have been targeted for therapeutic cerebral ablation, while also minimizing the risk of hemorrhage and infection from more invasive neurosurgical procedures. In a double-blinded, prospective, sham-controlled randomized controlled trial of MR-guided focused ultrasound thalamotomy for treatment of tremor-dominant PD, 62% of treated patients demonstrated improvement in tremor scores from baseline to 3 months postoperatively, as compared to 22% in the sham group. There has been only one open-label trial of MR-guided focused ultrasound subthalamotomy for patients with PD, demonstrating improvements of 71% for rigidity, 36% for akinesia, and 77% for tremor 6 months after treatment. Among the two open-label trials of MR-guided focused ultrasound pallidotomy for patients with PD, dyskinesia and overall motor scores improved up to 52% and 45% at 6 months postoperatively. Although MR-guided focused ultrasound thalamotomy is now approved by the U.S. Food and Drug Administration for treatment of parkinsonian tremor, additional high-quality randomized controlled trials are warranted and are underway to determine the safety and efficacy of MR-guided focused ultrasound subthalamotomy and pallidotomy for treatment of the cardinal features of PD. These studies will be paramount to aid clinicians to determine the ideal ablative target for individual patients. Additional work will be required to assess the durability of MR-guided focused ultrasound lesions, ideal timing of MR-guided focused ultrasound ablation in the course of PD, and the safety of performing bilateral lesions. © 2019 International Parkinson and Movement Disorder Society.

Zero TE MRI applications to transcranial MR-guided focused ultrasound: Patient screening and treatment efficiency estimation.

BACKGROUND: The high acoustic impedance of the skull limits the performance of transcranial magnetic resonance-guided focused ultrasound (tcMRgFUS) therapy. Subject suitability screening is based on skull parameters estimated from computed tomography (CT) scans. PURPOSE: To assess the feasibility of screening for tcMRgFUS based on zero echo time (ZTE) MRI, and to explore the influence of measurable skull parameters in treatment performance. STUDY TYPE: Retrospective. POPULATION: Sixteen patients treated with tcMRgFUS thalamotomy for tremor. SEQUENCE: ZTE on a 3.0T GE scanner. ASSESSMENT: Baseline CT and ZTE images were processed to extract skull measures associated with treatment success: skull density ratio (SDR), skull thickness, and angle of incidence. Eight new metrics were proposed. CT and ZTE-based measures were compared. Each subject's energy-temperature curve was processed to extract a global estimate of efficiency and a measure of nonlinearity. These parameters were then correlated with the skull measures. STATISTICAL TESTS: Linear regression analysis to compare ZTE vs. CT-based measures, measures vs. efficiency, and measures vs. nonlinearity. Paired t-test to assess nonlinearity. RESULTS: CT and ZTE-based measures were significantly correlated (P < 0.01). In particular, classical metrics were robustly replicated (P < 0.001). The energy-temperature curves showed a nonlinear (logarithmic) relationship (P < 0.01). This nonlinearity was greater for thicker skulls (P < 0.01). Efficiency was correlated with skull thickness (P < 0.001) and SDR (P < 0.05). DATA CONCLUSION: The feasibility of ZTE-based screening has been proven, potentially making it possible to avoid ionizing radiation and the extra imaging session required for CT. The characterization of the influence that skull properties have on tcMRgFUS may serve to develop patient-specific heating models, potentially improving control over the treatment outcome. The relationship of skull thickness with efficiency and nonlinearity empowers the role of this metric in the definition of such models. In addition, the lower association of SDR with the energy-temperature curves emphasizes the need of revisiting this metric. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;50:1583-1592.

Microstructural changes of the dentato-rubro-thalamic tract after transcranial MR guided focused ultrasound ablation of the posteroventral VIM in essential tremor.

Essential tremor is the most common movement disorder in adults. In patients who are not responsive to medical treatment, functional neurosurgery and, more recently, transcranial MR-guided focused ultrasound thalamotomy are considered effective therapeutic approaches. However, the structural brain changes following a thalamotomy that mediates the clinical improvement are still unclear. In here diffusion weighted images were acquired in a cohort of 24 essential tremor patients before and 3 months after unilateral transcranial MR-guided focused ultrasound thalamotomy targeting at the posteroventral part of the VIM. Microstructural changes along the DRTT were quantified by means of probabilistic tractography, and later related to the clinical improvement of the patients at 3-months and at 1-year after the intervention. In addition the changes along two neighboring tracts, that is, the corticospinal tract and the medial lemniscus, were assessed, as well as the relation between these changes and the presence of side effects. Thalamic lesions produced local and distant alterations along the trajectory of the DRTT, and each correlated with clinical improvement. Regarding side effects, gait imbalance after thalamotomy was associated with greater impact on the DRTT, whereas the presence of paresthesias was significantly related to a higher overlap between the lesion and the medial lemniscus. This work represents the largest series describing the microstructural changes following transcranial MR-guided focused ultrasound thalamotomy in essential tremor. These results suggest that clinical benefits are specific for the impact on the cerebello-thalamo-cortical pathway, thus reaffirming the potential of tractography to aid thalamotomy targeting.

Focused ultrasound subthalamotomy in patients with asymmetric Parkinson's disease: a pilot study.

BACKGROUND: Ablative neurosurgery has been used to treat Parkinson's disease for many decades. MRI-guided focused ultrasound allows focal lesions to be made in deep brain structures without skull incision. We investigated the safety and preliminary efficacy of unilateral subthalamotomy by focused ultrasound in Parkinson's disease. METHODS: This prospective, open-label pilot study was done at CINAC (Centro Integral de Neurociencias), University Hospital HM Puerta del Sur in Madrid, Spain. Eligible participants had Parkinson's disease with markedly asymmetric parkinsonism. Patients with severe dyskinesia, history of stereotactic surgery or brain haemorrhage, a diagnosis of an unstable cardiac or psychiatric disease, or a skull density ratio of 0·3 or less were excluded. Enrolled patients underwent focused ultrasound unilateral subthalamotomy. The subthalamic nucleus was targeted by means of brain images acquired with a 3-Tesla MRI apparatus. Several sonications above the definitive ablation temperature of 55°C were delivered and adjusted according to clinical response. The primary outcomes were safety and a change in the motor status of the treated hemibody as assessed with part III of the Movement Disorders Society-Unified Parkinson's Disease Rating Scale (MDS-UPDRS III) in both off-medication and on-medication states at 6 months. Adverse events were monitored up to 48 h after treatment and at scheduled clinic visits at 1, 3, and 6 months after treatment. The study is registered with ClinicalTrials.gov, number NCT02912871. FINDINGS: Between April 26 and June 14, 2016, ten patients with markedly asymmetric parkinsonism that was poorly controlled pharmacologically were enrolled for focused ultrasound unilateral subthalamotomy. By 6 months follow-up, 38 incidents of adverse events had been recorded, none of which were serious or severe. Seven adverse events were present at 6 months. Three of these adverse events were directly related to subthalamotomy: off-medication dyskinesia in the treated arm (one patient, almost resolved by 6 months); on-medication dyskinesia in the treated arm (one patient, resolved after levodopa dose reduction); and subjective speech disturbance (one patient). Four of the adverse events present at 6 months were related to medical management (anxiety and fatigue [one patient each] and weight gain [two patients]). The most frequent adverse events were transient gait ataxia (related to subthalamotomy, six patients), transient pin-site head pain (related to the head frame, six patients), and transient high blood pressure (during the procedure, five patients). Transient facial asymmetry (one patient) and moderate impulsivity (two patients) were also recorded. The mean MDS-UPDRS III score in the treated hemibody improved by 53% from baseline to 6 months in the off-medication state (16·6 [SD 2·9] vs 7·5 [3·9]) and by 47% in the on-medication state (11·9 [3·1] vs 5·8 [3·5]). INTERPRETATION: MRI-guided focused ultrasound unilateral subthalamotomy was well tolerated and seemed to improve motor features of Parkinson's disease in patients with noticeably asymmetric parkinsonism. Large randomised controlled trials are necessary to corroborate these preliminary findings and to assess the potential of such an approach to treat Parkinson's disease. FUNDING: Fundación de investigación HM Hospitales and Insightec.

Current applications and limitations of surgical treatments for movement disorders.

Functional neurosurgery for the treatment of both psychiatric and neurological disorders has been performed regularly since the 1940s. However, misuse in the early days and the appearance of effective medical treatments, such as levodopa and neuroleptic drugs, greatly reduced surgical approaches over several decades. The development of a comprehensive model of basal ganglia pathophysiology in the 1990s facilitated the resurgence of functional neurosurgery, mainly for the treatment of levodopa-related motor complications in Parkinson's disease. This led first to the re-emergence of posteroventral pallidotomy and subsequently to deep brain stimulation. Thirty years on from this turning point, we find ourselves looking at a new scenario. Although deep brain stimulation is accepted worldwide and technical advances continue to improve this therapy, new questions and challenges such as long-term benefits and optimal targeting have emerged. In addition, new nonincisional tools used to perform ablative treatments, such as high-intensity focused ultrasound and gamma-knife, are challenging classical reluctance to therapeutic lesioning, and it remains to be determined how these approaches will fit into the array of movement disorder treatments. This review discusses the current clinical state of the art of functional neurosurgery in the treatment of Parkinson's disease, tremor, and dystonia. © 2017 International Parkinson and Movement Disorder Society.

Biophysical methods to monitor structural aspects of the adenovirus infectious cycle.

In this chapter we compile a battery of biophysical and imaging methods suitable to investigate adenovirus structural stability, structure, and assembly. Some are standard methods with a long history of use in virology, such as embedding and sectioning of infected cells, negative staining, or immunoelectron microscopy, as well as extrinsic fluorescence. The newer cryo-electron microscopy technique, which combined with advanced image processing tools has recently yielded an atomic resolution picture of the complete virion, is also described. Finally, we detail the procedure for imaging and interacting with single adenovirus virions using the atomic force microscope in liquid conditions. We provide examples of the kind of data obtained with each technique.

The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis.

In eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

An off-target nucleostemin RNAi inhibits growth in human glioblastoma-derived cancer stem cells.

Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133(+) neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor.

Defining the specificity of cotranslationally acting chaperones by systematic analysis of mRNAs associated with ribosome-nascent chain complexes.

Polypeptides exiting the ribosome must fold and assemble in the crowded environment of the cell. Chaperones and other protein homeostasis factors interact with newly translated polypeptides to facilitate their folding and correct localization. Despite the extensive efforts, little is known about the specificity of the chaperones and other factors that bind nascent polypeptides. To address this question we present an approach that systematically identifies cotranslational chaperone substrates through the mRNAs associated with ribosome-nascent chain-chaperone complexes. We here focused on two Saccharomyces cerevisiae chaperones: the Signal Recognition Particle (SRP), which acts cotranslationally to target proteins to the ER, and the Nascent chain Associated Complex (NAC), whose function has been elusive. Our results provide new insights into SRP selectivity and reveal that NAC is a general cotranslational chaperone. We found surprising differential substrate specificity for the three subunits of NAC, which appear to recognize distinct features within nascent chains. Our results also revealed a partial overlap between the sets of nascent polypeptides that interact with NAC and SRP, respectively, and showed that NAC modulates SRP specificity and fidelity in vivo. These findings give us new insight into the dynamic interplay of chaperones acting on nascent chains. The strategy we used should be generally applicable to mapping the specificity, interplay, and dynamics of the cotranslational protein homeostasis network.

Structural mobility of the monomeric C-terminal domain of the HIV-1 capsid protein.

The capsid protein of HIV-1 (p24) (CA) forms the mature capsid of the human immunodeficiency virus. Capsid assembly involves hexamerization of the N-terminal domain and dimerization of the C-terminal domain of CA (CAC), and both domains constitute potential targets for anti-HIV therapy. CAC homodimerization occurs mainly through its second helix, and it is abolished when its sole tryptophan is mutated to alanine. This mutant, CACW40A, resembles a transient monomeric intermediate formed during dimerization. Its tertiary structure is similar to that of the subunits in the dimeric, non-mutated CAC, but the segment corresponding to the second helix samples different conformations. The present study comprises a comprehensive examination of the CACW40A internal dynamics. The results obtained, with movements sampling a wide time regime (from pico- to milliseconds), demonstrate the high flexibility of the whole monomeric protein. The conformational exchange phenomena on the micro-to-millisecond time scale suggest a role for internal motions in the monomer-monomer interactions and, thus, flexibility of the polypeptide chain is likely to contribute to the ability of the protein to adopt different conformational states, depending on the biological environment.

Flexibility in HIV-1 assembly subunits: solution structure of the monomeric C-terminal domain of the capsid protein.

The protein CA forms the mature capsid of human immunodeficiency virus. Hexamerization of the N-terminal domain and dimerization of the C-terminal domain, CAC, occur during capsid assembly, and both domains constitute potential targets for anti-HIV inhibitors. CAC homodimerization occurs mainly through its second helix, and is abolished when its sole tryptophan is mutated to alanine. Previous thermodynamic data obtained with the dimeric and monomeric forms of CAC indicate that the structure of the mutant resembles that of a monomeric intermediate found in the folding and association reactions of CAC. We have solved the three-dimensional structure in aqueous solution of the monomeric mutant. The structure is similar to that of the subunits in the dimeric, nonmutated CAC, except the segment corresponding to the second helix, which is highly dynamic. At the end of this region, the polypeptide chain is bent to bury several hydrophobic residues and, as a consequence, the last two helices are rotated 90 degrees when compared to their position in dimeric CAC. The previously obtained thermodynamic data are consistent with the determined structure of the monomeric mutant. This extraordinary ability of CAC to change its structure may contribute to the different modes of association of CA during HIV assembly, and should be taken into account in the design of new drugs against this virus.

Thermodynamic dissection of a low affinity protein-protein interface involved in human immunodeficiency virus assembly.

Homo-dimerization of the capsid protein CA of human immunodeficiency virus through its C-terminal domain constitutes an early crucial step in the virion assembly pathway and a potential target for antiviral inhibitors. We have truncated to alanine the 20 amino acid side chains per monomer that participate in intersubunit contacts at the CA dimer interface and analyzed their individual energetic contribution to protein association and stability. About half of the side chains in the contact epitope are critically involved in the energetic epitope as their truncation essentially prevented dimerization. However, dimerization affinity is kept low partly because of the presence of interfacial side chains whose individual truncation improves affinity by 2-20-fold. Many side chains at the interface are energetically important also for the folding of a monomeric intermediate and for its conformational rearrangement during dimerization. The thermodynamic description of this low affinity interface (dissociation constant of approximately 10 microm) was compared with those obtained for the other protein-protein interfaces, nearly all of them of much higher affinity, that have been systematically analyzed by mutation. The results reveal differences that may have been evolutionary selected and that may be exploited for the design of an effective interfacial inhibitor of human immunodeficiency virus assembly.