Biallelic Variants of MRPS36 Cause a New Form of Leigh Syndrome.

BACKGROUND: The MRPS36 gene encodes a recently identified component of the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the Krebs cycle catalyzing the oxidative decarboxylation of 2-oxoglutarate to succinyl-CoA. Defective OGDHC activity causes a clinically variable metabolic disorder characterized by global developmental delay, severe neurological impairment, liver failure, and early-onset lactic acidosis. METHODS: We investigated the molecular cause underlying Leigh syndrome with bilateral striatal necrosis in two siblings through exome sequencing. Functional studies included measurement of the OGDHC enzymatic activity and MRPS36 mRNA levels in fibroblasts, assessment of protein stability in transfected cells, and structural analysis. A literature review was performed to define the etiological and phenotypic spectrum of OGDHC deficiency. RESULTS: In the two affected brothers, exome sequencing identified a homozygous nonsense variant (c.283G>T, p.Glu95*) of MRPS36. The variant did not affect transcript processing and stability, nor protein levels, but resulted in a shorter protein lacking nine residues that contribute to the structural and functional organization of the OGDHC complex. OGDHC enzymatic activity was significantly reduced. The review of previously reported cases of OGDHC deficiency supports the association of this enzymatic defect with Leigh phenotypic spectrum and early-onset movement disorder. Slightly elevated plasma levels of glutamate and glutamine were observed in our and literature patients with OGDHC defect. CONCLUSIONS: Our findings point to MRPS36 as a new disease gene implicated in Leigh syndrome. The slight elevation of plasma levels of glutamate and glutamine observed in patients with OGDHC deficiency represents a candidate metabolic signature of this neurometabolic disorder. © 2024 International Parkinson and Movement Disorder Society.

A Recurrent Gain-of-Function Mutation in CLCN6, Encoding the ClC-6 Cl-/H+-Exchanger, Causes Early-Onset Neurodegeneration.

Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl-/H+ exchangers function as electric shunts for proton pumping and in luminal Cl- accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl-/H+-exchanger ClC-6. Whereas Clcn6-/- mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl-/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl- accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.

Mutations in KCNK4 that Affect Gating Cause a Recognizable Neurodevelopmental Syndrome.

Aberrant activation or inhibition of potassium (K+) currents across the plasma membrane of cells has been causally linked to altered neurotransmission, cardiac arrhythmias, endocrine dysfunction, and (more rarely) perturbed developmental processes. The K+ channel subfamily K member 4 (KCNK4), also known as TRAAK (TWIK-related arachidonic acid-stimulated K+ channel), belongs to the mechano-gated ion channels of the TRAAK/TREK subfamily of two-pore-domain (K2P) K+ channels. While K2P channels are well known to contribute to the resting membrane potential and cellular excitability, their involvement in pathophysiological processes remains largely uncharacterized. We report that de novo missense mutations in KCNK4 cause a recognizable syndrome with a distinctive facial gestalt, for which we propose the acronym FHEIG (facial dysmorphism, hypertrichosis, epilepsy, intellectual disability/developmental delay, and gingival overgrowth). Patch-clamp analyses documented a significant gain of function of the identified KCNK4 channel mutants basally and impaired sensitivity to mechanical stimulation and arachidonic acid. Co-expression experiments indicated a dominant behavior of the disease-causing mutations. Molecular dynamics simulations consistently indicated that mutations favor sealing of the lateral intramembrane fenestration that has been proposed to negatively control K+ flow by allowing lipid access to the central cavity of the channel. Overall, our findings illustrate the pleiotropic effect of dysregulated KCNK4 function and provide support to the hypothesis of a gating mechanism based on the lateral fenestrations of K2P channels.

Pathogenic PTPN11 variants involving the poly-glutamine Gln255 -Gln256 -Gln257 stretch highlight the relevance of helix B in SHP2's functional regulation.

Germline PTPN11 mutations cause Noonan syndrome (NS), the most common disorder among RASopathies. PTPN11 encodes SHP2, a protein tyrosine-phosphatase controlling signaling through the RAS-MAPK and PI3K-AKT pathways. Generally, NS-causing PTPN11 mutations are missense changes destabilizing the inactive conformation of the protein or enhancing its binding to signaling partners. Here, we report on two PTPN11 variants resulting in the deletion or duplication of one of three adjacent glutamine residues (Gln255 -to-Gln257 ). While p.(Gln257dup) caused a typical NS phenotype in carriers of a first family, p.(Gln257del) had incomplete penetrance in a second family. Missense mutations involving Gln256 had previously been reported in NS. This poly-glutamine stretch is located on helix B of the PTP domain, a region involved in stabilizing SHP2 in its autoinhibited state. Molecular dynamics simulations predicted that changes affecting this motif perturb the SHP2's catalytically inactive conformation and/or substrate recognition. Biochemical data showed that duplication and deletion of Gln257 variably enhance SHP2's catalytic activity, while missense changes involving Gln256 affect substrate specificity. Expression of mutants in HEK293T cells documented their activating role on MAPK signaling, uncoupling catalytic activity and modulation of intracellular signaling. These findings further document the relevance of helix B in the regulation of SHP2's function.

Structural Determinants of Phosphopeptide Binding to the N-Terminal Src Homology 2 Domain of the SHP2 Phosphatase.

SH2 domain-containing tyrosine phosphatase 2 (SHP2), encoded by PTPN11, plays a fundamental role in the modulation of several signaling pathways. Germline and somatic mutations in PTPN11 are associated with different rare diseases and hematologic malignancies, and recent studies have individuated SHP2 as a central node in oncogenesis and cancer drug resistance. The SHP2 structure includes two Src homology 2 domains (N-SH2 and C-SH2) followed by a catalytic protein tyrosine phosphatase (PTP) domain. Under basal conditions, the N-SH2 domain blocks the active site, inhibiting phosphatase activity. Association of the N-SH2 domain with binding partners containing short amino acid motifs comprising a phosphotyrosine residue (pY) leads to N-SH2/PTP dissociation and SHP2 activation. Considering the relevance of SHP2 in signaling and disease and the central role of the N-SH2 domain in its allosteric regulation mechanism, we performed microsecond-long molecular dynamics (MD) simulations of the N-SH2 domain complexed to 12 different peptides to define the structural and dynamical features determining the binding affinity and specificity of the domain. Phosphopeptide residues at position -2 to +5, with respect to pY, have significant interactions with the SH2 domain. In addition to the strong interaction of the pY residue with its conserved binding pocket, the complex is stabilized hydrophobically by insertion of residues +1, +3, and +5 in an apolar groove of the domain and interaction of residue -2 with both the pY and a protein surface residue. Additional interactions are provided by hydrogen bonds formed by the backbone of residues -1, +1, +2, and +4. Finally, negatively charged residues at positions +2 and +4 are involved in electrostatic interactions with two lysines (Lys89 and Lys91) specific for the SHP2 N-SH2 domain. Interestingly, the MD simulations illustrated a previously undescribed conformational flexibility of the domain, involving the core ß sheet and the loop that closes the pY binding pocket.

Design, Synthesis, Antibacterial Potential, and Structural Characterization of N-Acylated Derivatives of the Human Autophagy 16 Polypeptide.

A synthetic antimicrobial peptide library based on the human autophagy 16 polypeptide has been developed. Designed acetylated peptides bearing lipids of different chain lengths resulted in peptides with enhanced potency compared to the parent Atg16. A 21-residue fragment of Atg16 conjugated to 4-methylhexanoic acid (K30) emerged as the most potent antibacterial, with negligible hemolysis. Several studies, including microscopy, dye leakage, and ITC, were conducted to gain insight into the antibacterial mechanism of action of the peptide. Visual inspection using both SEM and TEM revealed the membranolytic effect of the peptide on bacterial cells. The selectivity of the peptide against bacterial cell membranes was also proven using dye leakage assays. ITC analysis revealed the exothermic nature of the binding interaction of the peptide to D8PG micelles. The three-dimensional solution NMR structure of K30 in complex with dioctanoylphosphatidylglycerol (D8PG) micelles revealed that the peptide adopts a helix-loop-helix structure in the presence of anionic membrane lipids mimicking bacterial membranes. Intermolecular NOEs between the peptide and lipid deciphered the location of the peptide in the bound state, which was subsequently supported by the paramagnetic relaxation enhancement (PRE) NMR experiment. Collectively, these results describe the structure-function relationship of the peptide in the bacterial membrane.

Targeting the Pentose Phosphate Pathway: Characterization of a New 6PGL Inhibitor.

Human African trypanosomiasis, or sleeping sickness, is a lethal disease caused by the protozoan parasite Trypanosoma brucei. However, although many efforts have been made to understand the biochemistry of this parasite, drug development has led to treatments that are of limited efficiency and of great toxicity. To develop new drugs, new targets must be identified, and among the several metabolic processes of trypanosomes that have been proposed as drug targets, carbohydrate metabolism (glycolysis and the pentose phosphate pathway (PPP)) appears as a promising one. As far as the PPP is concerned, a limited number of studies are related to the glucose-6-phosphate dehydrogenase. In this work, we have focused on the activity of the second PPP enzyme (6-phospho-gluconolactonase (6PGL)) that transforms 6-phosphogluconolactone into 6-phosphogluconic acid. A lactam analog of the natural substrate has been synthesized, and binding of the ligand to 6PGL has been investigated by NMR titration. The ability of this ligand to inhibit 6PGL has also been demonstrated using ultraviolet experiments, and protein-inhibitor interactions have been investigated through docking calculations and molecular dynamics simulations. In addition, a marginal inhibition of the third enzyme of the PPP (6-phosphogluconate dehydrogenase) was also demonstrated. Our results thus open new prospects for targeting T. brucei.

Molecular and cellular evidence of a direct interaction between the TRAF2 C-terminal domain and ganglioside GM1.

TNF receptor-associated factor 2 (TRAF2) is involved in different cellular processes including signal transduction and transcription regulation. We here provide evidence of a direct interaction between the TRAF domain of TRAF2 and the monosialotetrahexosylganglioside (GM1). Previously, we showed that the TRAF domain occurs mainly in a trimeric form in solution, but it can also exist as a stable monomer when in the nanomolar concentration range. Here, we report that the quaternary structure of the TRAF domain is also affected by pH changes, since a weakly acidic pH (5.5) favors the dissociation of the trimeric TRAF domain into stable monomers, as previously observed at neutral pH (7.6) with the diluted protein. The TRAF domain-GM1 binding was similar at pH 5.5 and 7.6, suggesting that GM1 interacts with both the trimeric and monomeric forms of the protein. However, only the monomeric protein appeared to cause membrane deformation and inward vesiculation in GM1-containing giant unilamellar vesicles (GUVs). The formation of complexes between GM1 and TRAF2, or its TRAF domain, was also observed in cultured human leukemic HAP1 cells expressing either the truncated TRAF domain or the endogenous full length TRAF2. The GM1-protein complexes were observed after treatment with tunicamycin and were more concentrated in cells undergoing apoptosis, a condition which is known to cause cytoplasm acidification. These findings open the avenue for future studies aimed at deciphering the physiopathological relevance of the TRAF domain-GM1 interaction.

Dominantly acting variants in vacuolar ATPase subunits impair lysosomal/autophagolysosome function causing a multisystemic disorder with neurocognitive impairment and multiple congenital anomalies.

The vacuolar H+-ATPase (V-ATPase) is a functionally conserved multimeric complex localized at the membranes of many organelles where its proton-pumping action is required for proper lumen acidification. The V-ATPase complex is composed of several subunits, some of which have been linked to human disease. We and others previously reported pathogenic dominantly acting variants in ATP6V1B2, the gene encoding the V1B2 subunit, as underlying a clinically variable phenotypic spectrum including dominant deafness-onychodystrophy (DDOD) syndrome, Zimmermann-Laband syndrome, and deafness, onychodystrophy, osteodystrophy, intellectual disability, and seizures (DOORS) syndrome. Here, we report on an individual with features fitting DOORS syndrome caused by dysregulated ATP6V1C1 function, expand the clinical features associated with ATP6V1B2 pathogenic variants, and provide evidence that these ATP6V1C1/ATP6V1B2 amino acid substitutions result in a gain-of-function mechanism upregulating V-ATPase function that drives increased lysosomal acidification. We demonstrate a disruptive effect of these ATP6V1B2/ATP6V1C1 variants on lysosomal morphology, localization and function, resulting in a defective autophagic flux and accumulation of lysosomal substrates. We also show that the upregulated V-ATPase function affects cilium biogenesis, further documenting pleiotropy. This work identifies ATP6V1C1 as a new gene associated with a neurodevelopmental phenotype resembling DOORS syndrome, documents the occurrence of a phenotypic continuum between ZLS, and DDOD and DOORS syndromes, and classify these conditions as lysosomal disorders.

Computational Evaluation of Peptide-Protein Binding Affinities: Application of Potential of Mean Force Calculations to SH2 Domains.

Many biological functions are mediated by protein-protein interactions (PPIs), often involving specific structural modules, such as SH2 domains. Inhibition of PPIs is a pharmaceutical strategy of growing importance. However, a major challenge in the design of PPI inhibitors is the large interface involved in these interactions, which, in many cases, makes inhibition by small organic molecules ineffective. Peptides, which cover a wide range of dimensions and can be opportunely designed to mimic protein sequences at PPI interfaces, represent a valuable alternative to small molecules. Computational techniques able to predict the binding affinity of peptides for the target domain or protein represent a crucial stage in the workflow for the design of peptide-based drugs. This chapter describes a protocol to obtain the potential of mean force (PMF) for peptide-SH2 domain binding, starting from umbrella sampling (US) molecular dynamics (MD) simulations. The PMF profiles can be effectively used to predict the relative standard binding free energies of different peptide sequences.

Fluorescence Anisotropy and Polarization in the Characterization of Biomolecular Association Processes and Their Application to Study SH2 Domain Binding Affinity.

Fluorescence anisotropy (or polarization) is a powerful technique to study biomolecular association processes, by following the rotational motions of one of the two partners in the interaction, labeled with a fluorophore. It can be used to determine dissociation constants in solution, down to nM values, and unlabeled ligands can be characterized, too, by using competition experiments. In this chapter, we introduce the basic principles of the technique, compare it with other experimental approaches, and discuss the experimental details with specific examples regarding SH2 domain/phosphopeptide association processes. The experimental protocols to be used in binding experiments and displacement studies are described, as well as the caveats to be considered in performing accurate measurements.

Isoamphipathic antibacterial molecules regulating activity and toxicity through positional isomerism.

Peptidomimetic antimicrobials exhibit a selective interaction with bacterial cells over mammalian cells once they have achieved an optimum amphiphilic balance (hydrophobicity/hydrophilicity) in the molecular architecture. To date, hydrophobicity and cationic charge have been considered the crucial parameters to attain such amphiphilic balance. However, optimization of these properties is not enough to circumvent unwanted toxicity towards mammalian cells. Hence, herein, we report new isoamphipathic antibacterial molecules (IAMs: 1-3) where positional isomerism was introduced as one of the guiding factors for molecular design. This class of molecules displayed good (MIC = 1-8 µg mL-1 or µM) to moderate [MIC = 32-64 µg mL-1 (32.2-64.4 µM)] antibacterial activity against multiple Gram-positive and Gram-negative bacteria. Positional isomerism showed a strong influence on regulating antibacterial activity and toxicity for ortho [IAM-1: MIC = 1-32 µg mL-1 (1-32.2 µM), HC50 = 650 µg mL-1 (654.6 µM)], meta [IAM-2: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 98 µg mL-1 (98.7 µM)] and para [IAM-3: MIC = 1-16 µg mL-1 (1-16.1 µM), HC50 = 160 µg mL-1 (161.1 µM)] isomers. Co-culture studies and investigation of membrane dynamics indicated that ortho isomer, IAM-1 exerted more selective activity towards bacterial over mammalian membranes, compared to meta and para isomers. Furthermore, the mechanism of action of the lead molecule (IAM-1) has been characterized through detailed molecular dynamics simulations. In addition, the lead molecule displayed substantial efficacy against dormant bacteria and mature biofilms, unlike conventional antibiotics. Importantly, IAM-1 exhibited moderate in vivo activity against MRSA wound infection in a murine model with no detectable dermal toxicity. Altogether, the report explored the design and development of isoamphipathic antibacterial molecules to establish the role of positional isomerism in achieving selective and potential antibacterial agents.

Insights into internal dynamics of 6-phosphogluconolactonase from Trypanosoma brucei studied by nuclear magnetic resonance and molecular dynamics.

Nuclear magnetic resonance is used to investigate the backbone dynamics in 6-phosphogluconolactonase from Trypanosoma brucei (Tb6PGL) with (holo-) and without (apo-) 6-phosphogluconic acid as ligand. Relaxation data were analyzed using the model-free approach and reduced spectral density mapping. Comparison of predictions, based on 77 ns molecular dynamics simulations, with the observed relaxation rates gives insight into dynamical properties of the protein and their alteration on ligand binding. Data indicate dynamics changes in the vicinity of the binding site. More interesting is the presence of perturbations located in remote regions of this well-structured globular protein in which no large-amplitude motions are involved. This suggests that delocalized changes in dynamics that occur upon binding could be a general feature of protein-target interactions.

ScrewFit: combining localization and description of protein secondary structure.

A new application of the ScrewFit algorithm [Kneller & Calligari (2006), Acta Cryst. D62, 302-311] is presented which adds the detection of protein secondary-structure elements to their detailed geometrical description in terms of a curve with intrinsic torsion. The extension is based on confidence and persistence criteria for the ScrewFit parameters which are established by analyzing the structural fluctuations of standard motifs in the SCOP fold classes. The agreement with the widely used DSSP method is comparable with the general consensus among other methods in the literature. This combination of secondary-structure detection and analysis is illustrated for the enzyme adenylate kinase.

Communication: a minimal model for the diffusion-relaxation backbone dynamics of proteins.

We present a model for the local diffusion-relaxation dynamics of the C(α)-atoms in proteins describing both the diffusive short-time dynamics and the asymptotic long-time relaxation of the position autocorrelation functions. The relaxation rate spectra of the latter are represented by shifted gamma distributions, where the standard gamma distribution describes anomalous slow relaxation in macromolecular systems of infinite size and the shift accounts for a smallest local relaxation rate in macromolecules of finite size. The resulting autocorrelation functions are analytic for any time t ≥ 0. Using results from a molecular dynamics simulation of lysozyme, we demonstrate that the model fits the position autocorrelation functions of the C(α)-atoms exceptionally well and reveals moreover a strong correlation between the residue's solvent-accessible surface and the fitted model parameters.

Least constraint approach to the extraction of internal motions from molecular dynamics trajectories of flexible macromolecules.

We propose a rigorous method for removing rigid-body motions from a given molecular dynamics trajectory of a flexible macromolecule. The method becomes exact in the limit of an infinitesimally small sampling step for the input trajectory. In a recent paper [G. Kneller, J. Chem. Phys. 128, 194101 (2008)], one of us showed that virtual internal atomic displacements for small time increments can be derived from Gauss' principle of least constraint, which leads to a rotational superposition problem for the atomic coordinates in two consecutive time frames of the input trajectory. Here, we demonstrate that the accumulation of these displacements in a molecular-fixed frame, which evolves in time according to the virtual rigid-body motions, leads to the desired trajectory for internal motions. The atomic coordinates in the input and output trajectory are related by a roto-translation, which guarantees that the internal energy of the molecule is left invariant. We present a convenient implementation of our method, in which the accumulation of the internal displacements is performed implicitly. Two numerical examples illustrate the difference to the classical approach for removing macromolecular rigid-body motions, which consists of aligning its configurations in the input trajectory with a fixed reference structure.

Discriminating between competing models for the allosteric regulation of oncogenic phosphatase SHP2 by characterizing its active state.

The Src-homology 2 domain containing phosphatase 2 (SHP2) plays a critical role in crucial signaling pathways and is involved in oncogenesis and in developmental disorders. Its structure includes two SH2 domains (N-SH2 and C-SH2), and a protein tyrosine phosphatase (PTP) domain. Under basal conditions, SHP2 is auto-inhibited, with the N-SH2 domain blocking the PTP active site. Activation involves a rearrangement of the domains that makes the catalytic site accessible, coupled to the association between the SH2 domains and cognate proteins containing phosphotyrosines. Several aspects of this transition are debated and competing mechanistic models have been proposed. A crystallographic structure of SHP2 in an active state has been reported (PDB code 6crf), but several lines of evidence suggests that it is not fully representative of the conformations populated in solution. To clarify the structural rearrangements involved in SHP2 activation, enhanced sampling simulations of the autoinhibited and active states have been performed, for wild type SHP2 and its pathogenic E76K variant. Our results demonstrate that the crystallographic conformation of the active state is unstable in solution, and multiple interdomain arrangements are populated, thus allowing association to bisphosphorylated sequences. Contrary to a recent proposal, activation is coupled to the conformational changes of the N-SH2 binding site, which is significantly more accessible in the active sate, rather than to the structure of the central ß-sheet of the domain. In this coupling, a previously undescribed role for the N-SH2 BG loop emerged.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

Molecular Investigation of SARS-CoV-2 Proteins and Their Interactions with Antiviral Drugs.

A new Coronavirus strain, named SARS-CoV-2, suddenly emerged in early December 2019. SARS-CoV-2 resulted in being dramatically infectious, with thousands of people infected. In this scenario, and without effective vaccines available, the importance of an immediate tool to support patients and against viral diffusion becomes evident. In this study, we exploit the molecular docking approach to analyze the affinity between different viral proteins and several inhibitors, originally developed for other viral infections. Our data show that, in some cases, a relevant binding can be detected. These findings support the hypothesis to develop new antiviral agents against COVID-19, on the basis of already established therapies.

Role of proteolytic enzymes in the COVID-19 infection and promising therapeutic approaches.

In the Fall of 2019 a sudden and dramatic outbreak of a pulmonary disease (Coronavirus Disease COVID-19), due to a new Coronavirus strain (i.e., SARS-CoV-2), emerged in the continental Chinese area of Wuhan and quickly diffused throughout the world, causing up to now several hundreds of thousand deaths. As for common viral infections, the crucial event for the viral life cycle is the entry of genetic material inside the host cell, realized by the spike protein of the virus through its binding to host receptors and its activation by host proteases; this is followed by translation of the viral RNA into a polyprotein, exploiting the host cell machinery. The production of individual mature viral proteins is pivotal for replication and release of new virions. Several proteolytic enzymes either of the host and of the virus act in a concerted fashion to regulate and coordinate specific steps of the viral replication and assembly, such as (i) the entry of the virus, (ii) the maturation of the polyprotein and (iii) the assembly of the secreted virions for further diffusion. Therefore, proteases involved in these three steps are important targets, envisaging that molecules which interfere with their activity are promising therapeutic compounds. In this review, we will survey what is known up to now on the role of specific proteolytic enzymes in these three steps and of most promising compounds designed to impair this vicious cycle.