Italian version of the anterior cruciate ligament-return to sport after injury scale (IT ACL-RSI): translation, cross-cultural adaptation, validation and ability to predict the return to sport at medium-term follow-up in a population of sport patients.

PURPOSE: The timing of psychological and physical recovery after anterior cruciate ligament reconstruction represents an open issue in current orthopedic practice. Several tools have been developed to evaluate these factors, with the most recent being represented by the anterior cruciate ligament (ACL) return to sport injury scale (ACL-RSI). The aims of this study were to provide a validated Italian translation of the ACL-RSI in a population of sport patients, and to identify a possible correlation of the ACL-RSI score with the return to sport (RTS) time and the level of sport participation in comparison to the pre-injury one. METHODS: The Italian translation and cultural adaptation of the scale were completed using a using the "translation-back translation" method. A total of 130 patients were enrolled and completed the study questionnaires 6 months after ACL reconstruction. Randomly, 65 of them were re-tested for the ACL-RSI within 2 weeks. The internal consistency, reliability, feasibility, and construct validity of the Italian version of ACL-RSI were assessed and compared to Italian version of the KOOS, the Lysholm Score, the AKPS and the IKDC subjective score. Responsiveness was tested comparing patients returning to sport at 6 and 12 months. The Tegner activity scale was collected at baseline, 6 and 12 months to identify the level of activity after return to sport, in relation to the ACL-RSI score. RESULTS: The Italian adaptation of the ACL-RSI demonstrated excellent internal consistency (Cronbach's alpha = 0.953), reliability (test-retest ICC = 0.916) and feasibility, with no ceiling or floor effect. Construct validity was confirmed by the moderate to strong correlation with all the other scales (p < 0.0001). Slight and non-significant higher ACL-RSI score was shown by patients returned to sport at 6 or 12 months after surgery. Nevertheless, the ACL-RSI score at 6 months was significantly different between patients who returned and those who did not returned to the same level of sport activity 12 months after the procedure. CONCLUSIONS: This study demonstrated that the Italian ACL-RSI is a reliable tool for evaluating the psychological readiness for return to sports of athletes who underwent ACL reconstruction, especially when collected at the end of the rehabilitation process. Since the IT ACL-RSI used in this study is a faithful translation of the original English version, this finding can be generalized to other cultural contexts and languages too. LEVEL OF EVIDENCE: Level II.

Targeting PDZ domains as potential treatment for viral infections, neurodegeneration and cancer.

The interaction between proteins is a fundamental event for cellular life that is generally mediated by specialized protein domains or modules. PDZ domains are the largest class of protein-protein interaction modules, involved in several cellular pathways such as signal transduction, cell-cell junctions, cell polarity and adhesion, and protein trafficking. Because of that, dysregulation of PDZ domain function often causes the onset of pathologies, thus making this family of domains an interesting pharmaceutical target. In this review article we provide an overview of the structural and functional features of PDZ domains and their involvement in the cellular and molecular pathways at the basis of different human pathologies. We also discuss some of the strategies that have been developed with the final goal to hijack or inhibit the interaction of PDZ domains with their ligands. Because of the generally low binding selectivity of PDZ domain and the scarce efficiency of small molecules in inhibiting PDZ binding, this task resulted particularly difficult to pursue and still demands increasing experimental efforts in order to become completely feasible and successful in vivo.

Probing the Effects of Local Frustration in the Folding of a Multidomain Protein.

Our current knowledge of protein folding is primarily based on experimental data obtained from isolated domains. In fact, because of their complexity, multidomain proteins have been elusive to the experimental characterization. Thus, the folding of a domain in isolation is generally assumed to resemble what should be observed for more complex structural architectures. Here we compared the folding mechanism of a protein domain in isolation and in the context of its supramodular multidomain structure. By carrying out an extensive mutational analysis we illustrate that while the early events of folding are malleable and influenced by the absence/presence of the neighboring structures, the late events appear to be more robust. These effects may be explained by analyzing the local frustration patterns of the domain, providing critical support for the funneled energy landscape theory of protein folding, and highlighting the role of protein frustration in sculpting the early events of the reaction.

Folding and Misfolding of a PDZ Tandem Repeat.

Although the vast majority of the human proteome is represented by multi-domain proteins, the study of multi-domain folding and misfolding is a relatively poorly explored field. The protein Whirlin is a multi-domain scaffolding protein expressed in the inner ear. It is characterized by the presence of tandem repeats of PDZ domains. The first two PDZ domains of Whirlin (PDZ1 and PDZ2 - namely P1P2) are structurally close and separated by a disordered short linker. We recently described the folding mechanism of the P1P2 tandem. The difference in thermodynamic stability of the two domains allowed us to selectively unfold one or both PDZ domains and to pinpoint the accumulation of a misfolded intermediate, which we demonstrated to retain physiological binding activity. In this work, we provide an extensive characterization of the folding and unfolding of P1P2. Based on the observed data, we describe an integrated kinetic analysis that satisfactorily fits the experiments and provides a valuable model to interpret multi-domain folding. The experimental and analytical approaches described in this study may be of general interest for the interpretation of complex multi-domain protein folding kinetics.

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery.

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted 'energetic coupling' describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein-ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

Targeting the Interaction between the SH3 Domain of Grb2 and Gab2.

Gab2 is a scaffolding protein, overexpressed in many types of cancers, that plays a key role in the formation of signaling complexes involved in cellular proliferation, migration, and differentiation. The interaction between Gab2 and the C-terminal SH3 domain of the protein Grb2 is crucial for the activation of the proliferation-signaling pathway Ras/Erk, thus representing a potential pharmacological target. In this study, we identified, by virtual screening, seven potential inhibitor molecules that were experimentally tested through kinetic and equilibrium binding experiments. One compound showed a remarkable effect in lowering the affinity of the C-SH3 domain for Gab2. This inhibitory effect was subsequently validated in cellula by using lung cancer cell lines A549 and H1299. Our results are discussed under the light of previous works on the C-SH3:Gab2 interaction.

Comparing the binding properties of peptides mimicking the Envelope protein of SARS-CoV and SARS-CoV-2 to the PDZ domain of the tight junction-associated PALS1 protein.

The Envelope protein (E) is one of the four structural proteins encoded by the genome of SARS-CoV and SARS-CoV-2 Coronaviruses. It is an integral membrane protein, highly expressed in the host cell, which is known to have an important role in Coronaviruses maturation, assembly and virulence. The E protein presents a PDZ-binding motif at its C-terminus. One of the key interactors of the E protein in the intracellular environment is the PDZ containing protein PALS1. This interaction is known to play a key role in the SARS-CoV pathology and suspected to affect the integrity of the lung epithelia. In this paper we measured and compared the affinity of peptides mimicking the E protein from SARS-CoV and SARS-CoV-2 for the PDZ domain of PALS1, through equilibrium and kinetic binding experiments. Our results support the hypothesis that the increased virulence of SARS-CoV-2 compared to SARS-CoV may rely on the increased affinity of its Envelope protein for PALS1.

Hidden kinetic traps in multidomain folding highlight the presence of a misfolded but functionally competent intermediate.

Although more than 75% of the proteome is composed of multidomain proteins, current knowledge of protein folding is based primarily on studies of isolated domains. In this work, we describe the folding mechanism of a multidomain tandem construct comprising two distinct covalently bound PDZ domains belonging to a protein called Whirlin, a scaffolding protein of the hearing apparatus. In particular, via a synergy between NMR and kinetic experiments, we demonstrate the presence of a misfolded intermediate that competes with productive folding. In agreement with the view that tandem domain swapping is a potential source of transient misfolding, we demonstrate that such a kinetic trap retains native-like functional activity, as shown by the preserved ability to bind its physiological ligand. Thus, despite the general knowledge that protein misfolding is intimately associated with dysfunction and diseases, we provide a direct example of a functionally competent misfolded state. Remarkably, a bioinformatics analysis of the amino acidic sequence of Whirlin from different species suggests that the tendency to perform tandem domain swapping between PDZ1 and PDZ2 is highly conserved, as demonstrated by their unexpectedly high sequence identity. On the basis of these observations, we discuss on a possible physiological role of such misfolded intermediate.

Demonstration of Binding Induced Structural Plasticity in a SH2 Domain.

SH2 domains are common protein interaction domains able to recognize short aminoacidic sequences presenting a phosphorylated tyrosine (pY). In spite of their fundamental importance for cell physiology there is a lack of information about the mechanism by which these domains recognize and bind their natural ligands. The N-terminal SH2 (N-SH2) domain of PI3K mediates the interaction with different scaffolding proteins and is known to recognize a specific pY-X-X-M consensus sequence. These interactions are at the cross roads of different molecular pathways and play a key role for cell development and division. By combining mutagenesis, chemical kinetics and NMR, here we provide a complete characterization of the interaction between N-SH2 and a peptide mimicking the scaffolding protein Gab2. Our results highlight that N-SH2 is characterized by a remarkable structural plasticity, with the binding reaction being mediated by a diffused structural region and not solely by the residues located in the binding pocket. Furthermore, the analysis of kinetic data allow us to pinpoint an allosteric network involving residues far from the binding pocket involved in specificity. Results are discussed on the light of previous works on the binding properties of SH2 domains.

Tendon morphological changes after a prolonged ski race can be detected by ultrasound echo intensity.

BACKGROUND: Ultrasound imaging techniques have been used to assess the characteristics of skeletal muscles and tendons. Such techniques (gray scale analysis) allow qualitative evaluation and have been used recently to assess the internal structure of muscles and tendons by computer-aided gray scale analysis. We hypothesized that changes in the internal structure of the Achilles and patellar tendons after a ski mountaineering race competition could be detected with ultrasound. METHODS: Twenty athletes were recruited during the 19th Millet Tour du Rutor extreme, a three-day ski mountaineering competition. Ultrasound measurements of the Achilles and patellar tendons were carried out before the first race and immediately after each of the three competition days. Tendon thickness, cross-sectional area (CSA), and ultrasound gray scale analysis were calculated. RESULTS: Significant differences (p < 0.05) were observed between the pre- and post-race measurements for the Achilles tendon thickness and CSA, while no significant differences were noted for the patellar tendon thickness and CSA. However, gray scale analysis of both the Achilles and patellar tendons showed significantly higher post-race values, than the pre-race values (p < 0.05). CONCLUSIONS: Achilles and patellar tendons of healthy athletes are highly responsive to an acute increase in mechanical load. Those changes can be detected from classical (thickness and CSA) and innovative (gray scale) ultrasound-based parameters. TRIAL REGISTRATION: This study was approved by the Azienda USL Valle d'Aosta Ethics Committee (protocol no. 23/03/2018.0026243.I).

Understanding the Mechanism of Recognition of Gab2 by the N-SH2 Domain of SHP2.

Gab2 is a scaffold protein with a crucial role in colocalizing signaling proteins and it is involved in the regulation of several important molecular pathways. SHP2 is a protein phosphatase that binds, through its two SH2 domains, specific consensus sequences presenting a phosphorylated tyrosine located on the disordered tail of Gab2. To shed light on the details of such a fundamental interaction for the physiology of the cell, we present a complete mutational analysis of the kinetics of binding between the N-SH2 domain of SHP2 and a peptide mimicking a specific region of Gab2. By analyzing kinetic data, we determined structural features of the transition state of the N-SH2 domain binding to Gab2, highlighting a remarkable cooperativity of the binding reaction. Furthermore, comparison of these data with ones previously obtained for another SH2 domain suggests the presence of underlying general features characterizing the binding process of SH2 domains. Data are discussed under the light of previous works on SH2 domains.

Understanding the Binding Induced Folding of Intrinsically Disordered Proteins by Protein Engineering: Caveats and Pitfalls.

Many proteins lack a well-defined three-dimensional structure in isolation. These proteins, typically denoted as intrinsically disordered proteins (IDPs), may display a characteristic disorder-to-order transition when binding their physiological partner(s). From an experimental perspective, it is of great importance to establish the general grounds to understand how such folding processes may be explored. Here we discuss the caveats and the pitfalls arising when applying to IDPs one of the key techniques to characterize the folding of globular proteins, the Φ value analysis. This method is based on measurements of the free energy changes of transition and native states upon conservative, non-disrupting, mutations. On the basis of available data, we reinforce the validity of Φ value analysis in the study of IDPs and suggest future experiments to further validate this powerful experimental method.

Templated folding of intrinsically disordered proteins.

Much of our current knowledge of biological chemistry is founded in the structure-function relationship, whereby sequence determines structure that determines function. Thus, the discovery that a large fraction of the proteome is intrinsically disordered, while being functional, has revolutionized our understanding of proteins and raised new and interesting questions. Many intrinsically disordered proteins (IDPs) have been determined to undergo a disorder-to-order transition when recognizing their physiological partners, suggesting that their mechanisms of folding are intrinsically different from those observed in globular proteins. However, IDPs also follow some of the classic paradigms established for globular proteins, pointing to important similarities in their behavior. In this review, we compare and contrast the folding mechanisms of globular proteins with the emerging features of binding-induced folding of intrinsically disordered proteins. Specifically, whereas disorder-to-order transitions of intrinsically disordered proteins appear to follow rules of globular protein folding, such as the cooperative nature of the reaction, their folding pathways are remarkably more malleable, due to the heterogeneous nature of their folding nuclei, as probed by analysis of linear free-energy relationship plots. These insights have led to a new model for the disorder-to-order transition in IDPs termed "templated folding," whereby the binding partner dictates distinct structural transitions en route to product, while ensuring a cooperative folding.

Comparison of the effectiveness of manual massage, long-wave diathermy, and sham long-wave diathermy for the management of delayed-onset muscle soreness: a randomized controlled trial.

BACKGROUND: Delayed-onset muscle soreness (DOMS) is a specific symptom that typically arises after unaccustomed eccentric muscular effort. It increases typically 24-72 h post-exercise and can affect physical performance. The pathophysiology of DOMS remains unclear, although it seems to be related to the remodeling phase of myofibrils. Different types of treatments have been proposed to minimize DOMS after exercise; however, no clear gold standard treatment exists. Among the most popular and easy-to-apply treatments, manual massage is often performed by clinicians and has been documented to be effective in reducing symptoms. For several years, long-wave diathermy (LWD) has been performed to manage musculoskeletal complaints, such as DOMS; however, no studies have reported its efficacy thus far.This study aimed to compare the clinical effectiveness of LWD, sham LWD, and manual massage in participants with lower limb DOMS. METHODS: Participants with lower limb DOMS were included in the study. They were randomly assigned to undergo real LWD, sham LWD, or manual massage. The Numeric Pain Rating Scale (NPRS) score was the primary outcome, and the Patient Global Impression of Change (PGIC) Scale score was the secondary outcome. Outcomes were collected before and immediately after the treatment. Analysis of variance was performed to compare the post-treatment NPRS value variability among the groups and to compare the pre- and post-treatment NPRS differences among the groups. RESULTS: No clinically relevant differences were observed regarding the NPRS value variability among real LWD, sham LWD and manual massage groups. Differences were observed in the PGIC Scale scores. CONCLUSIONS: Future studies are needed to have a better understanding about the treatment of DOMS in clinical practice. TRIAL REGISTRATION: The trial was registered on 29th February 2016 in ClinicalTrials.gov (NCT02693678).

Unveiling the Molecular Basis of the Noonan Syndrome-Causing Mutation T42A of SHP2.

Noonan syndrome (NS) is a genetic disorder caused by the hyperactivation of the RAS-MAPK molecular pathway. About 50% of NS cases are caused by mutations affecting the SHP2 protein, a multi-domain phosphatase with a fundamental role in the regulation of the RAS-MAPK pathway. Most NS-causing mutations influence the stability of the inactive form of SHP2. However, one NS-causing mutation, namely T42A, occurs in the binding pocket of the N-SH2 domain of the protein. Here, we present a quantitative characterization of the effect of the T42A mutation on the binding of the N-terminal SH2 domain of SHP2 with a peptide mimicking Gab2, a fundamental interaction that triggers the activation of the phosphatase in the cellular environment. Our results show that whilst the T42A mutation does not affect the association rate constant with the ligand, it causes a dramatic increase of the affinity for Gab2. This effect is due to a remarkable decrease of the microscopic dissociation rate constant of over two orders of magnitudes. In an effort to investigate the molecular basis of the T42A mutation in causing Noonan syndrome, we also compare the experimental results with a more conservative variant, T42S. Our findings are discussed in the context of the structural data available on SHP2.

The Effect of Proline cis-trans Isomerization on the Folding of the C-Terminal SH2 Domain from p85.

SH2 domains are protein domains that modulate protein-protein interactions through a specific interaction with sequences containing phosphorylated tyrosines. In this work, we analyze the folding pathway of the C-terminal SH2 domain of the p85 regulatory subunit of the protein PI3K, which presents a proline residue in a cis configuration in the loop between the ßE and ßF strands. By employing single and double jump folding and unfolding experiments, we demonstrate the presence of an on-pathway intermediate that transiently accumulates during (un)folding. By comparing the kinetics of folding of the wild-type protein to that of a site-directed variant of C-SH2 in which the proline was replaced with an alanine, we demonstrate that this intermediate is dictated by the peptidyl prolyl cis-trans isomerization. The results are discussed in the light of previous work on the effect of peptidyl prolyl cis-trans isomerization on folding events.

Binding induced folding: Lessons from the kinetics of interaction between NTAIL and XD.

Intrinsically Disordered Proteins (IDPs) are a class of protein that exert their function despite lacking a well-defined three-dimensional structure, which is sometimes achieved only upon binding to their natural ligands. This feature implies the folding of IDPs to be generally coupled with a binding event, representing an interesting challenge for kinetic studies. In this review, we recapitulate some of the most important findings of IDPs binding-induced folding mechanisms obtained by analyzing their binding kinetics. Furthermore, by focusing on the interaction between the Measles virus NTAIL protein, a prototypical IDP, and its physiological partner, the X domain, we recapitulate the major theoretical and experimental approaches that were used to describe binding induced folding.

Structural characterization of an on-pathway intermediate and transition state in the folding of the N-terminal SH2 domain from SHP2.

Src Homology 2 (SH2) domains are a class of protein domains that present a conserved three-dimensional structure and possess a crucial role in mediating protein-protein interactions. Despite their importance and abundance in the proteome, knowledge about the folding properties of SH2 domain is limited. Here we present an extensive mutational analysis (Φ value analysis) of the folding pathway of the N-SH2 domain of the Src homology region 2 domain-containing phosphatase-2 (SHP2) protein, a 104 residues domain that presents the classical SH2 domain fold (two α-helices flanking a central ß-sheet composed of 3-5 antiparallel ß-strands), with a fundamental role in mediating the interaction of SHP2 with its substrates and triggering key metabolic pathways in the cell. By analysing folding kinetic data we demonstrated that the folding pathway of N-SH2 presents an obligatory on-pathway intermediate that accumulates during the folding reaction. The production of 24 conservative site-directed variants allowed us to perform a Φ value analysis, by which we could fully characterize the intermediate and the transition state native-like interactions, providing a detailed quantitative analysis of the folding pathway of N-SH2. Results highlight the presence of a hydrophobic nucleus that stabilizes the intermediate, leading to a higher degree of native-like interactions in the transition state. Data are discussed and compared with previous works on SH2 domains.

The kinetics of folding of the NSH2 domain from p85.

SH2 domains are protein domains that mediate protein-protein interaction through the recognition and binding of specific sequences containing phosphorylated tyrosines. The p85 protein is the regulatory subunit of the heterodimeric enzyme PI3K, an important enzyme involved in several molecular pathways. In this work we characterize the folding kinetics of the NSH2 domain of p85. Our data clearly reveal peculiar folding kinetics, characterized by an apparent mismatch between the observed folding and unfolding kinetics. Taking advantage of double mixing stopped flow experiments and site directed mutagenesis we demonstrate that such behavior is due to the cis/trans isomerization of the peptide bond between D73 and P74, being in a cis conformation in the native protein. Our data are discussed in comparison with previous works on the folding of other SH2 domains.

Investigating the Molecular Basis of the Aggregation Propensity of the Pathological D76N Mutant of Beta-2 Microglobulin: Role of the Denatured State.

Beta-2 microglobulin (ß2m) is a protein responsible for a pathologic condition, known as dialysis-related amyloidosis (DRA), caused by its aggregation and subsequent amyloid formation. A naturally occurring mutation of ß2m, D76N, presents a higher amyloidogenic propensity compared to the wild type counterpart. Since the three-dimensional structure of the protein is essentially unaffected by the mutation, the increased aggregation propensity of D76N has been generally ascribed to its lower thermodynamic stability and increased dynamics. In this study we compare the equilibrium unfolding and the aggregation propensity of wild type ß2m and D76N variant at different experimental conditions. Our data revealed a surprising effect of the D76N mutation in the residual structure of the denatured state, which appears less compact than that of the wild type protein. A careful investigation of the structural malleability of the denatured state of wild type ß2m and D76N pinpoint a clear role of the denatured state in triggering the amyloidogenic propensity of the protein. The experimental results are discussed in the light of the previous work on ß2m and its role in disease.