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Matrix metalloproteinases (MMPs) are significant drivers of many diseases, including cancer, and are established targets for drug development. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous MMP inhibitors and are being pursued for the development of anti-MMP therapeutics. TIMPs possess many attractive properties for drug candidates, such as complete MMP inhibition, low toxicity, low immunogenicity, and high tissue permeability. However, a major challenge with TIMPs is their rapid clearance from the bloodstream due to their small size. This study explores a method for extending the plasma half-life of the N-terminal domain of TIMP2 (N-TIMP2) by appending it with a long, intrinsically unfolded tail containing Pro, Ala, and Thr (PATylation). We designed and produced two PATylated N-TIMP2 constructs with tail lengths of 100 and 200 amino acids (N-TIMP2-PAT100 and N-TIMP2-PAT200). Both constructs demonstrated higher apparent molecular weights and retained high inhibitory activity against MMP-9. N-TIMP2-PAT200 significantly increased plasma half-life in mice compared to the non-PATylated variant, enhancing its therapeutic potential. PATylation offers distinct advantages for half-life extension, such as fully genetic encoding, monodispersion, and biodegradability. It can be easily applied to N-TIMP2 variants engineered for high affinity and selectivity toward individual MMPs, creating promising candidates for drug development against MMP-related diseases.

Mutations in the PINK1 and PRKN genes are the most frequent genetic cause of early-onset Parkinson disease. The pathogenic p.R275W substitution in PRKN is the most frequent substitution observed in patients, and thus far has been characterized mostly through overexpression models that suggest a possible gain of toxic misfunction. However, its effects under endogenous conditions are largely unknown. We used patient fibroblasts, isogenic neurons, and post-mortem human brain samples from carriers with and without PRKN p.R275W to assess functional impact. Immunoblot analysis and immunofluorescence were used to study mitophagy activation, and mitophagy execution was analyzed by flow cytometry of the reporter mitoKeima. The functional analysis was accompanied by structural investigation of PRKN p.R275W. We observed lower PRKN protein in fibroblasts with compound heterozygous p.R275W mutations. Isogenic neurons showed an allele-dose dependent decrease in PRKN protein. Lower PRKN protein levels were accompanied by diminished phosphorylated ubiquitin and decreased MFN2 modification. Mitochondrial degradation was also allele-dose dependently impaired. Consistently, PRKN protein levels were drastically reduced in human brain samples from p.R275W carriers. Finally, structural simulations showed significant changes in the closed form of PRKN p.R275W. Our data suggest that under endogenous conditions the p.R275W mutation results in a loss-of-function by destabilizing PRKN.

Complete loss-of-function mutations in the PRKN gene are a major cause of early-onset Parkinson’s disease (PD). PRKN encodes the Parkin protein, an E3 ubiquitin ligase that works in conjunction with the ubiquitin kinase PINK1 in a distinct quality control pathway to tag damaged mitochondria for autophagic clearance, i.e., mitophagy. According to previous structural investigations, Parkin protein is typically kept in an inactive conformation via several intramolecular, auto-inhibitory interactions. Here, we performed molecular dynamics simulations (MDS) to provide insights into conformational changes occurring during the de-repression of Parkin and the gain of catalytic activity. We analyzed four different Parkin-activating mutations that are predicted to disrupt certain aspects of its auto-inhibition. All four variants showed greater conformational motions compared to wild-type protein, as well as differences in distances between domain interfaces and solvent-accessible surface area, which are thought to play critical roles as Parkin gains catalytic activity. Our findings reveal that the studied variants exert a notable influence on Parkin activation as they alter the opening of its closed inactive structure, a finding that is supported by recent structure- and cell-based studies. These findings not only helped further characterize the hyperactive variants but overall improved our understanding of Parkin’s catalytic activity and nominated targets within Parkin’s structure for potential therapeutic designs.

(1) Background: Mutations in NFκB1, a transcriptional regulator of immunomodulating proteins, are a known cause of inborn errors of immunity. Our proband is a 22-year-old male with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Genetic studies identified a novel, single-point mutation variant in NFκB1, c. T638A p. V213E. (2) Methods: Next-generation panel sequencing of the patient uncovered a novel single-point mutation in the NFκB1 gene that was modeled using the I-TASSER homology-modeling software, and molecular dynamics were assessed using the YASARA2 software (version 20.14.24). (3) Results: This variant replaces valine with glutamic acid at position 213 in the NFκB1 sequence. Molecular modeling and molecular dynamic studies showed altered dynamics in and around the rel homology domain, ankyrin regions, and death domain of the protein. We postulate that these changes alter overall protein function. (4) Conclusions: This case suggests the pathogenicity of a novel variant using protein-modeling techniques and molecular dynamic simulations.

Abstract BACKGROUND Mutations of NFkB1 are a known cause of inborn errors of immunity resulting in immunodeficiency due to the role of NFkB1 as a transcriptional regulator of immunomodulating proteins. We present a patient with a diagnosis of common variable immunodeficiency (CVID), cytopenias with massive splenomegaly, and nodular regenerative hyperplasia of the liver. Our proband, a 22-year-old male, presents with a clinical picture of immunodeficiency, and has a maternal cousin with a similar gene mutation but differing phenotype. Next generation panel sequencing (NGPS) was utilized to discover a novel, single point mutation variant in NFkB1, V213E, that is present in our proband. METHODS The novel single-point mutation V213E in the NFkB gene was modeled in-silico using the I-TASSER homology modeling software. Molecular dynamics simulations of both the wild type and variant mutant form fitted to accommodate the novel mutation were conducted and analyzed to assess for possible pathogenicity of the novel mutation. RESULTS This variant replaces Valine with Glutamic Acid at position 213 in the transcription regulator’s sequence. We perform molecular modeling to analyze the proteomic impact and difference in molecular dynamics (MDS) which depicted suppressed dynamics in the ankyrin region and death domain of the protein complex. Analysis of the proband’s family showed no significant family history except for the maternal cousin with a mutation in NFkB1. CONCLUSION This case both highlights the heterogeneity in phenotype of NFkB1 pathogenic variants as shown in this patient, full penetrance of the variant mutation, and suggests the pathogenicity of a new variant through protein modeling techniques and molecular dynamic simulations.

Abstract Matrix metalloproteinases (MMPs) regulate the degradation of extracellular matrix (ECM) components in biological processes. MMP activity is controlled by natural tissue inhibitors of metalloproteinases (TIMPs) that non-selectively inhibit the function of multiple MMPs via interaction with the MMPs' Zn2+-containing catalytic pocket. Recent studies suggest that TIMPs engineered to confer MMP specificity could be exploited for therapeutic purposes, but obtaining specific TIMP-2 inhibitors has proved to be challenging. Here, in an effort to improve MMP specificity, we incorporated the metal-binding non-canonical amino acids (NCAAs), 3,4-dihydroxyphenylalanine (L-DOPA) and (8-hydroxyquinolin-3-yl)alanine (HqAla), into the MMP-inhibitory N-terminal domain of TIMP2 (N-TIMP2) at selected positions that interact with the catalytic Zn2+ ion (S2, S69, A70, L100) or with a structural Ca2+ ion (Y36). Evaluation of the inhibitory potency of the NCAA-containing variants towards MMP-2, MMP-9 and MMP-14 in vitro revealed that most showed a significant loss of inhibitory activity towards MMP-14, but not towards MMP-2 and MMP-9, resulting in increased specificity towards the latter proteases. Substitutions at S69 conferred the best improvement in selectivity for both L-DOPA and HqAla variants. Molecular modeling revealed how MMP-2 and MMP-9 are better able to accommodate the bulky NCAA substituents at the intermolecular interface with N-TIMP2. The models also showed that, rather than coordinating to Zn2+, the NCAA side chains formed stabilizing polar interactions at the intermolecular interface with MMP-2 and MMP-9. The findings illustrate how incorporation of NCAAs can be used to probe and exploit differential tolerance for substitution within closely related protein-protein complexes to achieve improved specificity.

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that function to mediate tissue remodeling; however, they are also implicated in pathological conditions such as cancer, inflammatory and degenerative diseases. There are 23 human MMPs and significant sequence and structural homology exists among them and with other metalloproteinases. This represents a substantial challenge in developing therapeutically viable selective inhibitors due to off‐target effects. Tissue inhibitors of metalloproteinases (TIMPs) are a family of four endogenous protein protease inhibitors that regulate MMP activity. Our lab and others have utilized TIMPs as scaffolds, engineering them to selectively target specific MMPs for potential therapeutic applications. We previously employed a yeast surface display platform to identify TIMP‐1 variants possessing enhanced MMP‐3 affinity. In the current study, we used structures of MMP‐3 co‐crystallized with either WT TIMP‐1 or an engineered TIMP‐1 variant as seeds for molecular dynamics simulations. Simulations were also conducted on unbound WT and engineered TIMP‐1 to examine MMP‐independent dynamics that distinguish the engineered variant. The simulation data were analyzed with root‐mean‐square deviation/fluctuation, time‐dependent secondary structure, temporally resolved hydrogen bonding, conformational clustering, and dynamical network algorithms to discern structural and dynamical determinants of the increased affinity. Key observations that demonstrate the increased effectiveness of engineered TIMP‐1 as an MMP‐3 inhibitor were secondary structural changes in the MMP‐binding loops in proximity to mutations, attenuated global and mutation‐adjacent dynamics, decreased conformational dynamics, and increased persistence of MMP‐3‐compatible conformations. There were also indications of greater interdomain cooperativity within the engineered TIMP‐1 variant: increased interdomain hydrogen bonding, coordinated motions of the N‐terminal region with the C‐terminal domain, and increased intercommunity information flow/motion transfer. From these observations, we hypothesize the increased interdomain interactions represent a mechanism of dynamical stabilization such that mainly cooperative motion between domains presides, rather than random independent motions. Our findings suggest propagated motions between domains may influence the MMP affinity of TIMPs. Furthermore, these data suggest future strategies to design next‐generation mutant TIMP libraries by targeting regions dynamically coupled to the MMP binding loops for variation, thus exploiting natural protein dynamics to achieve desired selectivity.

The PRSS23 gene, encoding putative serine protease 23, has been associated with several diseases by correlative gene expression studies and genetic‐based comparative models. Our group has recently developed data implicating the expression of this gene as a potential driver of ovarian cancer malignancy. However, there have been no studies defining PRSS23 protein structure or demonstrating its hypothesized proteolytic function. We aim to elucidate PRSS23 protein structure and enzymatic activity. The first step to achieve this goal is to develop a robust expression system and purification protocol for recombinant PRSS23 protein. We hypothesize that developing a methodology to produce PRSS23 recombinant protein will enable us to determine its crystal structure, evaluate its biological and enzyme function, and ultimately target the enzyme therapeutically. We strategized to maximize chances of producing PRSS23 in its native form and with high yield by testing multiple systems and host species: human, yeast, and bacterial. We anticipate, based on the protein sequence, that PRSS23 is endogenously secreted and may be activated in the secretory pathway by furin, releasing the C‐terminal catalytic domain from a smaller N‐terminal domain. The protein is also predicted to be glycosylated. We generated expression plasmids for full‐length PRSS23, codon‐optimized for expression in human and Pichia pastoris expression systems, incorporating a His‐tag for detection and purification. In the human HEK 293F and HEK 293T cell systems, despite optimizing transfection protocols and testing multiple expression constructs, secretion signals, and promoters, the expression level remained very low and was judged unlikely to produce sufficient protein for structural and functional studies. Integration of codon‐optimized PRSS23 into the genome of P. pastoris produced integrants showing a spectrum of expression levels from very low to moderate. Experiments are ongoing to optimize expression conditions of the best clone and develop a purification workflow involving ammonium sulfate precipitation, immobilized nickel ion chromatography, and additional chromatographic steps as needed. In parallel, we cloned the catalytic domain of PRSS23, appropriately codon‐optimized and also containing a C‐terminal His‐tag, into an expression vector for Escherichia coli. High levels of protein were produced as insoluble inclusion bodies, and a protocol was developed for nickel ion affinity purification under denaturing conditions. Experiments are ongoing to identify suitable conditions for in vitro refolding of the PRSS23 catalytic domain. Additionally, we are evaluating a maltose binding protein fusion with the PRSS23 catalytic domain as an alternative strategy to improve soluble protein production in E. coli. Significant quantities of pure protein will be needed to enable crystal structure determination and enzymatic activity elucidation, to unravel the biological mechanism of action and identify endogenous substrates, and potentially lead to new therapeutic strategies targeting PRSS23. The expression systems and purification workflows developed here will be a step forward toward these long‐term goals.

Serine protease inhibitors of the Kunitz‐bovine pancreatic trypsin inhibitor (BPTI) family, such as tissue factor pathway inhibitor‐1 Kunitz domain 1 ( KD1 TFPI1) and amyloid precursor protein inhibitor (APPI), are biological regulators of proteolysis and serve as important scaffolds for developing therapeutics. Mesotrypsin is a unique human protease with cancer‐promoting properties which rapidly cleaves and inactivates serine protease inhibitors; because of this, it is a challenging therapeutic target and an excellent model for developing therapeutic inhibitors with improved proteolytic resistance. We hypothesize that stabilization of the Kunitz inhibitor binding loop by disulfide bond anchoring to the scaffold can reduce proteolytic susceptibility and improve binding efficacy. Here we have engineered a new disulfide bond into both KD1 TFPI1 and an APPI variant previously engineered for mesotrypsin affinity. The new disulfide bond anchors the binding loop to the scaffold, resulting in reduced mesotrypsin proteolytic cleavage by 17‐fold and 3‐fold, respectively. To gain insight into the structural basis for improved proteolytic stability, we crystallized mutant KD1 TFPI1 and APPI with mesotrypsin and solved these novel structures. We then performed all‐atom molecular dynamics simulations using these mesotrypsin‐mutant inhibitor complexes compared to models lacking the engineered disulfide bond. Our simulation results support our hypothesis that the cause of decreased proteolysis rates is dampened conformational fluctuations in the binding loop and minimized motion at the enzyme‐inhibitor interface. We also performed simulations using unbound inhibitors to show that stabilization of the binding loop to states similar to the bound configuration correlates with stronger binding affinity. Our disulfide engineering approach is potentially a useful tool in biopharmaceutical development for mesotrypsin, other serine proteases, and possibly for other enzyme targets. Support or Funding Information NIH grant R01CA154387 to E.S.R., European Research Council grant 336041 to N.P., US‐Israel Binational Science Foundation grant BSF2015134 to E.S.R. and N.P. Xray diffraction data were measured at the Berkeley Center for Structural Biology at the Advanced Light Source and at the European Synchrotron Radiation Facility. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .