Proteolytic Processing Research Articles

MET Oncogene Enhances Pro-Migratory Functions by Counteracting NMDAR2B Cleavage.

The involvement of the N-methyl-D-aspartate receptor (NMDAR), a glutamate-gated ion channel, in promoting the invasive growth of cancer cells is an area of ongoing investigation. Our previous findings revealed a physical interaction between NMDAR and MET, the hepatocyte growth factor (HGF) receptor. However, the molecular mechanisms underlying this NMDAR/MET interaction remain unclear. In this study, we demonstrate that the NMDAR2B subunit undergoes proteolytic processing, resulting in a low-molecular-weight form of 100 kDa. Interestingly, when the NMDAR2B and MET constructs were co-transfected, the full-size high-molecular-weight NMDAR2B form of 160 kDa was predominantly observed. The protection of NMDAR2B from cleavage was dependent on the kinase activity of MET. We provide the following evidence that MET opposes the autophagic lysosomal proteolysis of NMDAR2B: (i) MET decreased the protein levels of lysosomal cathepsins; (ii) treatment with either an inhibitor of autophagosome formation or the fusion of the autophagosome and lysosome elevated the proportion of the NMDAR2B protein's uncleaved form; (iii) a specific mTOR inhibitor hindered the anti-autophagic effect of MET. Finally, we demonstrate that MET coopts NMDAR2B to augment cell migration. This implies that MET harnesses the functionality of NMDAR2B to enhance the ability of cancer cells to migrate.

ATXN2 is a target of N-terminal proteolysis.

Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder caused by the expansion of the poly-glutamine (polyQ) tract of Ataxin-2 (ATXN2). Other polyQ-containing proteins such as ATXN7 and huntingtin are associated with the development of neurodegenerative diseases when their N-terminal polyQ domains are expanded. Furthermore, they undergo proteolytic processing events that produce N-terminal fragments that include the polyQ stretch, which are implicated in pathogenesis. Interestingly, N-terminal ATXN2 fragments were reported in a brain extract from a SCA2 patient, but it is currently unknown whether an expanded polyQ domain contributes to ATXN2 proteolytic susceptibility. Here, we used transient expression in HEK293 cells to determine whether ATXN2 is a target for specific N-terminal proteolysis. We found that ATXN2 proteins with either normal or expanded polyQ stretches undergo proteolytic cleavage releasing an N-terminal polyQ-containing fragment. We identified a short amino acid sequence downstream of the polyQ domain that is necessary for N-terminal cleavage of full-length ATXN2 and sufficient to induce proteolysis of a heterologous protein. However, this sequence is not required for cleavage of a short ATXN2 isoform produced from an alternative start codon located just upstream of the CAG repeats encoding the polyQ domain. Our study extends our understanding of ATXN2 posttranslational regulation by revealing that this protein can be the target of specific proteolytic cleavage events releasing polyQ-containing products that are modulated by the N-terminal domain of ATXN2. N-terminal ATXN2 proteolysis of expanded polyQ domains might contribute to SCA2 pathology, as observed in other neurodegenerative disorders caused by polyQ domain expansion.

The role of plant proteases in the response of plants to abiotic stress factors

The primary function of proteolytic enzymes is the hydrolysis of peptide bonds. Enzymes responsible for catalyzing this reaction are commonly referred to as proteases. The vast majority of these enzymes belong to the class of hydrolases and operate in aqueous environments. However, there is a distinct group of proteases known as intramembrane proteases, which are integral membrane proteins capable of cleaving peptide bonds in the hydrophobic environment of biological membranes. Proteolysis serves several essential functions in plant cells, ranging from the degradation of damaged and unnecessary proteins to the removal of non-functional protein aggregates. It plays a role in the quality control system for proteins and even releases transcription factors from membrane proteins. Proteolytic processes are indispensable at every stage of plant development and allow for the modification of the cell’s protein composition based on developmental needs and environmental requirements. Proteases are also enzymatic components of the cell that facilitate the efficient regeneration of many key metabolic pathways, such as photosynthesis and respiration. The response of plant cells and the entire plant organism to various biotic and abiotic stresses often requires the remodeling of metabolic pathways, the regeneration of key enzymatic complexes, or changes in the protein profile. By participating in all these processes, proteases constitute a crucial element of the cellular response to environmental stresses. The aim of this work is to review the role that individual proteases play in the response of plant cells to abiotic stress factors, such as drought, salinity, cold, temperature, and light.

Proteolytic Changes During Ripening Of Kučki Cheese

Summary Production of various traditional dairy products, particularly cheese, has a long history in Montenegro. White-brined cheeses are the most well-known. One of them is Kučki cheese, with distinct flavor and higher level of proteolysis. The aim of this research was to analyze the proteolysis process during ripening of Kučki cheese. The cheese is produced using traditional technology. FTIR Spectrophotometry was used to calculate the amount of total protein in cheese (IDF141C: 2000). The degree of proteolysis was determined by SDS PAGE (Laemmli, 1970). The cheese was examined for their water-soluble nitrogen (WSN) content using Kuchroo and Fox’s method from 1982, as well as their 5% phosphotungstic acid soluble nitrogen (PTAN) content using Stadhouser’s method from 1960. The results were expressed as percentages of WSN and PTAN of the total nitrogen matter (WSN/TN and PTAN/TN), as well as PTAN as a percentage of WSN (PTAN/WSN). All analyses were done on 10th, 20th and 30th day of cheese ripening. Four samples of cheese were analyzed for each ripening period. The content of proteins increased during the ripening period. The parameters that determine the process of proteolysis increased during the first 30 days of ripening. The WSN/TN ranged from 13.33 to 44.32%. Also, PTAN/WSN varied from 3.79 to 21.57%. The initial results show that uneven ripening conditions have a direct impact on how proteolytic changes develop throughout ripening. Due to the absence of established cheese-making procedures, Kučki cheese has a considerable degree of heterogeneity in their protein content and proteolysis parameters. However, it is not possible to strictly define the optimal values of the degree of ripening for Kučki cheese. In order to reach certain conclusions, it is necessary to carry out further studies focusing on analyzing a larger number of samples.

Loss of Complement Factor I (Cfi) a Negative Regulator of Complement Cascade Activity Exacerbates JAK2V617F-Dependent Phenotype

Background: The current evidence suggests that inflammatory phenotype contributes to the pathogenesis of myeloproliferative neoplasms (MPNs). While our understanding of complement cascade contributions to inflammation-mediated pathologies has expanded, role this cascade plays in etiology of myeloid malignancies has not been detailed. Previously, we uncovered an inactivating RNSV in complement factor I (CFI G119R), an inhibitor of the complement cascade, in 20% of PMF patients (n=10) using WGS, and in a separate patient cohort (n=10) we observed an increase in CH50 levels (n=3) and decrease in C3 levels (n=3) (Oakes et al., 2021, Blood, 138:1472).These findings prompted us to generate murine models allowing assessment of the Cfi loss-driven complement overactivity effects on hematopoiesis and development of MPNs. Methods: A conventional Cfi knockout (KO) mouse model was generated by targeting exon 2 of the Cfi gene using CRISPR/Cas9. Inactivation of one or two copies of Cfi was confirmed by genotyping and total loss (Cfi KO) or Cfi deficiency (Cfi HET) were confirmed in peripheral blood (PB) plasma. Complete blood counts (CBC) and the frequencies of granulocytes, B cells, plasmocytes, T cells, macrophages/monocytes, platelets, erythroid cells in the bone marrow were longitudinally assessed by flow cytometry for 40 weeks in Cfi WT, HET and KO mice. Cfi HET animals were crossed with JAK2V617F(fl/+);Mx1-cre(+) mice (obtained from crossing B6.Cg-Tg( Mx1- cre+ 1)Cgn/J; # 003556, JAX) and JAK2V617F expressing (B6N.129S6(SJL)- Jak2 tm1.2Ble/AmlyJ #031658, JAX) to generate Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre(+/-) (CFI/JAK2) strain. Complete blood counts and survival were monitored for Cfi(+/+)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI WT/JAK2 HET) Cfi(+/-)/JAK2V617F(fl/+);Tg(Mx1-cre)(+/-) (CFI HET/JAK2 HET) or Cfi(-/-)/JAK2V617F(fl/+); Tg(Mx1-cre)(+/-) (CFI KO/JAK2 HET) mice. Results:Cfi HET and KO mice were fertile and viable. Cohort of Cfi WT n=13, Het n=12, KO n=13 mice was subjected to analyses. Genotypes were confirmed via PCR and western blot. Flow cytometry demonstrated a copy loss dependent decrease in total T-cells (CD3+) and increase in mature resting B-cells (CD19+/B220+). CBC demonstrated a copy loss dependent decrease in both monocyte (p adj. KO vs. WT 0.002) and granulocyte percentage (p adj. KO vs. WT 0.003), and increase in lymphocyte percentage (p adj. KO vs. WT 0.001). In PB plasma we detected increased C3 activation in KO mice compared to WT and Het, demonstrated as decreased C3 alpha and presence of inactivated C3 due to increased proteolytical processing of C3 complex. Proteolytic cleavage of CFB compared to Cfi WT and Het was also noted in Cfi KO animals. A significant decrease in survival probability of CFI HET/JAK2 HET vs. CFI WT/JAK2 HET (p=0.03), CFI KO/JAK2 HET vs. CFI WT/JAK2 HET (p=0.003), and CFI HET/JAK2 HET vs. CFI KO/JAK2 HET (p=0.05) averaging 92 days for Cfi HET/JAK2 HET, 67 days for Cfi KO/JAK2 HET vs. 98 days for Cfi WT/JAK2 HET was noted (Fig. A). A granulocyte and monocyte percentage of CFI HET/JAK2 HET was increased as compared to CFI WT/JAK2 HET mice (Fig. B). Conclusions:An overactivation of complement cascade at the C3 level is noted in Cfi deficient animals, that is phenotypically linked to an increase in frequency of B cells. These findings may suggest involvement of Cfi directly, and/or in conjunction with generally overactive complement, in B-cell maturation and function. The absence of one copy of Cfi in JAK2V617F mice shows more severe MPN disease progression resulting in markedly decreased survival, linked to increased frequencies of monocytes and granulocytes and markedly decreased lymphocytes. In sum, these data indicate that increased complement activity may increase JAK2V617F-dependent phenotype severity. In depth mechanistic studies detailing observed phenotype are warranted.

Antibody-mediated spike activation promotes cell-cell transmission of SARS-CoV-2.

The COVID pandemic fueled by emerging SARS-CoV-2 new variants of concern remains a major global health concern, and the constantly emerging mutations present challenges to current therapeutics. The spike glycoprotein is not only essential for the initial viral entry, but is also responsible for the transmission of SARS-CoV-2 components via syncytia formation. Spike-mediated cell-cell transmission is strongly resistant to extracellular therapeutic and convalescent antibodies via an unknown mechanism. Here, we describe the antibody-mediated spike activation and syncytia formation on cells displaying the viral spike. We found that soluble antibodies against receptor binding motif (RBM) are capable of inducing the proteolytic processing of spike at both the S1/S2 and S2' cleavage sites, hence triggering ACE2-independent cell-cell fusion. Mechanistically, antibody-induced cell-cell fusion requires the shedding of S1 and exposure of the fusion peptide at the cell surface. By inhibiting S1/S2 proteolysis, we demonstrated that cell-cell fusion mediated by spike can be re-sensitized towards antibody neutralization in vitro. Lastly, we showed that cytopathic effect mediated by authentic SARS-CoV-2 infection remain unaffected by the addition of extracellular neutralization antibodies. Hence, these results unveil a novel mode of antibody evasion and provide insights for antibody selection and drug design strategies targeting the SARS-CoV-2 infected cells.

Neuron-specific gene NSG1 binds to and positively regulates sortilin ectodomain shedding via a metalloproteinase-dependent mechanism

Increasing evidence suggests that aberrant regulation of sortilin ectodomain shedding can contribute to amyloid-β pathology and frontotemporal dementia, although the mechanism by which this occurs has not been elucidated. Here we probed for novel binding partners of sortilin using multiple and complementary approaches and identified two proteins of the neuron-specific gene (NSG) family, NSG1 and NSG2, that physically interact and colocalize with sortilin. We show both NSG1 and NSG2 induce subcellular redistribution of sortilin to NSG1- and NSG2-enriched compartments. However, using cell surface biotinylation we found only NSG1 reduced sortilin cell surface expression, which caused significant reductions in uptake of progranulin (PGRN), a molecular determinant for frontotemporal dementia. In contrast, we demonstrate NSG2 has no effect on sortilin cell surface abundance or PGRN uptake, suggesting specificity for NSG1 in the regulation of sortilin cell surface expression. Using metalloproteinase inhibitors and A Disintegrin And Metalloproteinase 10 (ADAM10) knockout cells, we further show that NSG1-dependent reduction of cell surface sortilin occurred via proteolytic processing by ADAM10 with a concomitant increase in shedding of sortilin ectodomain to the extracellular space. This represents a novel regulatory mechanism for sortilin ectodomain shedding that is regulated in a neuron-specific manner. Furthermore, this finding has implications for the development of strategies for brain-specific regulation of sortilin and possibly sortilin-driven pathologies.

Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation

AbstractThe NLRP3 inflammasome is a cytosolic protein complex that regulates innate immune signaling in response to diverse pathogenic insults through the proteolytic processing and secretion of pro‐inflammatory cytokines such as IL‐1β. Hyperactivation of NLRP3 inflammasome signaling is implicated in the onset and pathogenesis of numerous diseases, motivating the discovery of new strategies to suppress NLRP3 inflammasome activity. We sought to define the potential for the proteostasis regulator AA147 to inhibit the assembly and activation of the NLRP3 inflammasome. AA147 is a pro‐drug that is metabolically converted to a reactive metabolite at the endoplasmic reticulum (ER) membrane to covalently modify ER‐localized proteins such as protein disulfide isomerases (PDIs). We show that AA147 inhibits NLRP3 inflammasome activity in monocytes and monocyte‐derived macrophages through a mechanism involving impaired assembly of the active inflammasome complex. This inhibition is mediated through AA147‐dependent covalent modification of PDIA1. Genetic depletion or treatment with other highly selective PDIA1 inhibitors similarly blocks NLRP3 inflammasome assembly and activation. Our results identify PDIA1 as a potential therapeutic target to mitigate NLRP3 inflammasome‐mediated pro‐inflammatory signaling implicated in etiologically diverse diseases.

Role of the NLRP1 inflammasome in skin cancer and inflammatory skin diseases.

Inflammasomes are cytoplasmic protein complexes that play a crucial role in protecting the host against pathogenic and sterile stressors by initiating inflammation. Upon activation, these complexes directly regulate the proteolytic processing and activation of proinflammatory cytokines interleukin (IL)-1β and IL-18 to induce a potent inflammatory response, and induce a programmed form of cell death called pyroptosis to expose intracellular pathogens to the surveillance of the immune system, thus perpetuating inflammation. There are various types of inflammasome complexes, with the NLRP1 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-1) inflammasome being the first one identified and currently recognized as the predominant inflammasome sensor protein in human keratinocytes. Human NLRP1 exhibits a unique domain structure, containing both an N-terminal pyrin (PYD) domain and an effector C-terminal caspase recruitment domain (CARD). It can be activated by diverse stimuli, such as viruses, ultraviolet B radiation and ribotoxic stress responses. Specific mutations in NLRP1 or related genes have been associated with rare monogenic skin disorders, such as multiple self-healing palmoplantar carcinoma; familial keratosis lichenoides chronica; autoinflammation with arthritis and dyskeratosis; and dipeptidyl peptidase 9 deficiency. Recent research breakthroughs have also highlighted the involvement of dysfunctions in the NLRP1 pathway in a handful of seemingly unrelated dermatological conditions. These range from monogenic autoinflammatory diseases to polygenic autoimmune diseases such as vitiligo, psoriasis, atopic dermatitis and skin cancer, including squamous cell carcinoma, melanoma and Kaposi sarcoma. Additionally, emerging evidence implicates NLRP1 in systemic lupus erythematosus, pemphigus vulgaris, Addison disease, Papillon-Lefèvre syndrome and leprosy. The aim of this review is to shed light on the implications of pathological dysregulation of the NLRP1 inflammasome in skin diseases and investigate the potential rationale for targeting this pathway as a future therapeutic approach.

Loss of UCP2 causes mitochondrial fragmentation by OMA1-dependent proteolytic processing of OPA1 in podocytes.

Mitochondrial dysfunction plays an important role in the onset and progression of podocyte injury and proteinuria. However, the process by which the change in the podocyte mitochondria occurs is not well understood. Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier protein, which is located in the mitochondrial inner membrane. Here, we reported that mice with podocyte-specific Ucp2 deficiency developed podocytopathy with proteinuria with aging. Furthermore, those mice exhibited increased proteinuria in experimental models evoked by Adriamycin. Our findings suggest that UCP2 mediates mitochondrial dysfunction by regulating mitochondrial dynamic balance. Ucp2-deleted podocytes exhibited increased mitochondrial fission and deficient in ATP production. Mechanistically, opacity protein 1 (OPA1), a key protein in fusion of mitochondrial inner membrane, was regulated by UCP2. Ucp2 deficiency promoted proteolysis of OPA1 by activation OMA1 which belongs to mitochondrial inner membrane zinc metalloprotease. Those finding demonstrate the role of UCP2 in mitochondrial dynamics in podocytes and provide new insights into pathogenesis associated with podocyte injury and proteinuria.

Amino acid editing of NFE2L1 by PNGase causes abnormal mobility on SDS-PAGE

NFE2L1 (also known as NRF1) is a member of the nuclear erythroid 2-like family of transcription factors and is critical for counteracting various types of cellular stress such as oxidative, proteotoxic or metabolic stress. This unique transcription factor is also known to undergo changes, including post-translational modifications, limited proteolysis or translocation into the nucleus, before it exerts full transcriptional activity. As a result, there are various molecular forms with distinct sizes for this protein, while the precise nature of each form remains elusive. In this study, the N-glycosylated status of NFE2L1 in cells was examined. The findings revealed that when NFE2L1 was deglycosylated by PNGase F, the size-shift on SDS-PAGE was minimal. This was in contrast to deglycosylation by Endo H, which resulted in a clear size-shift, even though N-linked GlcNAc residues remained on the protein. It was found that this unusual behavior of PNGase-deglycosylated NFE2L1 was dependent on the conversion of the glycosylated-Asn to Asp, resulting in the introduction of more negative charges into the core peptide of NFE2L1. We also demonstrate that NGLY1-mediated deglycosylation and DDI2-mediated proteolytic processing of NFE2L1 are not strictly ordered reactions. Our study will allow us to better understand the precise structures as well as biochemical properties of the various forms of NFE2L1.

Fibrocystin/Polyductin releases a C-terminal fragment that translocates into mitochondria and suppresses cystogenesis

Fibrocystin/Polyductin (FPC), encoded by PKHD1, is associated with autosomal recessive polycystic kidney disease (ARPKD), yet its precise role in cystogenesis remains unclear. Here we show that FPC undergoes complex proteolytic processing in developing kidneys, generating three soluble C-terminal fragments (ICDs). Notably, ICD15, contains a novel mitochondrial targeting sequence at its N-terminus, facilitating its translocation into mitochondria. This enhances mitochondrial respiration in renal epithelial cells, partially restoring impaired mitochondrial function caused by FPC loss. FPC inactivation leads to abnormal ultrastructural morphology of mitochondria in kidney tubules without cyst formation. Moreover, FPC inactivation significantly exacerbates renal cystogenesis and triggers severe pancreatic cystogenesis in a Pkd1 mouse mutant Pkd1V/V in which cleavage of Pkd1-encoded Polycystin-1 at the GPCR Proteolysis Site is blocked. Deleting ICD15 enhances renal cystogenesis without inducing pancreatic cysts in Pkd1V/V mice. These findings reveal a direct link between FPC and a mitochondrial pathway through ICD15 cleavage, crucial for cystogenesis mechanisms.

A unique borrelial protein facilitates microbial immune evasion.

Lyme disease is a major tick-borne infection caused by a bacterial pathogen called Borrelia burgdorferi, which is transmitted by ticks and affects hundreds of thousands of people every year. These bacterial pathogens are distinct from other genera of microbes because of their distinct features and ability to transmit a multi-system infection to a range of vertebrates, including humans. Progress in understanding the infection biology of Lyme disease, and thus advancements towards its prevention, are hindered by an incomplete understanding of the microbiology of B. burgdorferi, partly due to the occurrence of many unique borrelial proteins that are structurally unrelated to proteins of known functions yet are indispensable for pathogen survival. We herein report the use of diverse technologies to examine the structure and function of a unique B. burgdorferi protein, annotated as BB0238-an essential virulence determinant. We show that the protein is structurally organized into two distinct domains, is involved in multiplex protein-protein interactions, and facilitates tick-to-mouse pathogen transmission by aiding microbial evasion of early host cellular immunity. We believe that our findings will further enrich our understanding of the microbiology of B. burgdorferi, potentially impacting the future development of novel prevention strategies against a widespread tick-transmitted infection.

Unveiling the peptidome diversity of Lachesismuta snake venom: Discovery of novel fragments of metalloproteinase, l-amino acid oxidase, and bradykinin potentiating peptides

Snake venoms are known to be major sources of peptides with different pharmacological properties. In this study, we comprehensively explored the venom peptidomes of three specimens of Lachesismuta, the largest venomous snake in South America, using mass spectrometry techniques. The analysis revealed 19 main chromatographic peaks common to all specimens. A total of 151 peptides were identified, including 69 from a metalloproteinase, 58 from the BPP-CNP precursor, and 24 from a l-amino acid oxidase. To our knowledge, 126 of these peptides were reported for the first time in this work, including a new SVMP-derived peptide fragment, Lm-10a. Our findings highlight the dynamic nature of toxin maturation in snake venoms, driven by proteolytic processing, post-translational modifications, and cryptide formation.

Impact of mutations on the plant-based production of recombinant SARS-CoV-2 RBDs.

Subunit vaccines based on recombinant viral antigens are valuable interventions to fight existing and evolving viruses and can be produced at large-scale in plant-based expression systems. The recombinant viral antigens are often derived from glycosylated envelope proteins of the virus and glycosylation plays an important role for the immunogenicity by shielding protein epitopes. The receptor-binding domain (RBD) of the SARS-CoV-2 spike is a principal target for vaccine development and has been produced in plants, but the yields of recombinant RBD variants were low and the role of the N-glycosylation in RBD from different SARS-CoV-2 variants of concern is less studied. Here, we investigated the expression and glycosylation of six different RBD variants transiently expressed in leaves of Nicotiana benthamiana. All of the purified RBD variants were functional in terms of receptor binding and displayed almost full N-glycan occupancy at both glycosylation sites with predominately complex N-glycans. Despite the high structural sequence conservation of the RBD variants, we detected a variation in yield which can be attributed to lower expression and differences in unintentional proteolytic processing of the C-terminal polyhistidine tag used for purification. Glycoengineering towards a human-type complex N-glycan profile with core α1,6-fucose, showed that the reactivity of the neutralizing antibody S309 differs depending on the N-glycan profile and the RBD variant.

Tissue inhibitors of metalloproteinases are proteolytic targets of matrix metalloproteinase 9

Extracellular proteolysis and turnover are core processes of tissue homeostasis. The predominant matrix-degrading enzymes are members of the Matrix Metalloproteinase (MMP) family. MMPs extensively degrade core matrix components in addition to processing a range of other factors in the extracellular, plasma membrane, and intracellular compartments. The proteolytic activity of MMPs is modulated by the Tissue Inhibitors of Metalloproteinases (TIMPs), a family of four multi-functional matrisome proteins with extensively characterized MMP inhibitory functions. Thus, a well-regulated balance between MMP activity and TIMP levels has been described as critical for healthy tissue homeostasis, and this balance can be chronically disturbed in pathological processes. The relationship between MMPs and TIMPs is complex and lacks the constraints of a typical enzyme-inhibitor relationship due to secondary interactions between various MMPs (specifically gelatinases) and TIMP family members. We illustrate a new complexity in this system by describing how MMP9 can cleave members of the TIMP family when in molar excess. Proteolytic processing of TIMPs can generate functionally altered peptides with potentially novel attributes. We demonstrate here that all TIMPs are cleaved at their C-terminal tails by a molar excess of MMP9. This processing removes the N-glycosylation site for TIMP3 and prevents the TIMP2 interaction with latent proMMP2, a prerequisite for cell surface MMP14-mediated activation of proMMP2. TIMP2/4 are further cleaved producing ∼14 kDa N-terminal proteins linked to a smaller C-terminal domain through residual disulfide bridges. These cleaved TIMP2/4 complexes show perturbed MMP inhibitory activity, illustrating that MMP9 may bear a particularly prominent influence upon the TIMP:MMP balance in tissues.

Axon-Autonomous Effects of the Amyloid Precursor Protein Intracellular Domain (AICD) on Kinase Signaling and Fast Axonal Transport.

The amyloid precursor protein (APP) is a key molecular component of Alzheimer's disease (AD) pathogenesis. Proteolytic APP processing generates various cleavage products, including extracellular amyloid beta (Aβ) and the cytoplasmic APP intracellular domain (AICD). Although the role of AICD in the activation of kinase signaling pathways is well established in the context of full-length APP, little is known about intracellular effects of the AICD fragment, particularly within discrete neuronal compartments. Deficits in fast axonal transport (FAT) and axonopathy documented in AD-affected neurons prompted us to evaluate potential axon-autonomous effects of the AICD fragment for the first time. Vesicle motility assays using the isolated squid axoplasm preparation revealed inhibition of FAT by AICD. Biochemical experiments linked this effect to aberrant activation of selected axonal kinases and heightened phosphorylation of the anterograde motor protein conventional kinesin, consistent with precedents showing phosphorylation-dependent regulation of motors proteins powering FAT. Pharmacological inhibitors of these kinases alleviated the AICD inhibitory effect on FAT. Deletion experiments indicated this effect requires a sequence encompassing the NPTY motif in AICD and interacting axonal proteins containing a phosphotyrosine-binding domain. Collectively, these results provide a proof of principle for axon-specific effects of AICD, further suggesting a potential mechanistic framework linking alterations in APP processing, FAT deficits, and axonal pathology in AD.

AAV-mediated skeletal muscle specific irisin expression does not contribute to weight loss in mice

Obesity, a chronic disease, significantly increases the risk of various diseases, including diabetes, cardiovascular diseases, and cancers. Exercise is crucial for weight management not only through energy expenditure by muscle activity but also through stimulating the secretion of myokines, which affect various tissues. Irisin, derived from the proteolytic processing of fibronectin type III domain-containing protein 5 (Fndc5), is a well-studied myokine with beneficial effects on metabolism. This study explored the feasibility of adeno-associated virus (AAV)-mediated Fndc5 gene therapy to treat obesity in a mouse model using the AAV-DIO system to express Fndc5 specifically in skeletal muscle, and investigated its anti-obesity effect. Although Fndc5 was specifically expressed in the muscle, no significant impact on body weight under normal chow or high-fat diets was observed, and no change in thermogenic gene expression in inguinal white adipose tissue was detected. Notably, Fndc5 transduction did affect bone metabolism, consistent with previous reports. These findings suggest that AAV-mediated Fndc5 gene therapy may not be an efficient strategy for obesity, contrary to our expectations. Further research is needed to elucidate the complex mechanisms involved in irisin's role in obesity and related disorders.

Masking the transmembrane region of the amyloid β precursor protein as a safe means to lower amyloid β production.

Reducing brain levels of both soluble and insoluble forms of amyloid beta (Aβ) remains the primary goal of most therapies that target Alzheimer's disease (AD). However, no treatment has so far resulted in patient benefit, and clinical trials of the most promising drug candidates have generally failed due to significant adverse effects. This highlights the need for safer and more selective ways to target and modulate Aβ biogenesis. Peptide technology has advanced to allow reliable synthesis, purification, and delivery of once-challenging hydrophobic sequences. This is opening up new routes to target membrane processes associated with disease. Here we deploy a combination of atomic detail molecular dynamics (MD) simulations, living-cell Förster resonance energy transfer (FRET), and in vitro assays to elucidate the atomic-detail dynamics, molecular mechanisms, and cellular activity and selectivity of a membrane-active peptide that targets the Aβ precursor protein (APP). We demonstrate that Aβ biogenesis can be downregulated selectively using an APP occlusion peptide (APPOP). APPOP inhibits Aβ production in a dose-dependent manner, with a mean inhibitory concentration (IC50) of 450nM toward exogenous APP and 50nM toward endogenous APP in primary rat cortical neuronal cultures. APPOP does not impact the γ-secretase cleavage of Notch-1, or exhibit toxicity toward cultured primary rat neurons, suggesting that it selectively shields APP from proteolysis. Drugs targeting AD need to be given early and for very long periods to prevent the onset of clinical symptoms. This necessitates being able to target Aβ production precisely and without affecting the activity of key cellular enzymes such as γ-secretase for other substrates. Peptides offer a powerful way for targeting key pathways precisely, thereby reducing the risk of adverse effects. Here we show that protecting APP from proteolytic processing offers a promising route to safely and specifically lower Aβ burden. In particular, we show that the amyloid pathway can be targeted directly and specificically. This reduces the risk of off-target effects and paves the way for a safe prophylactic treatment.

Chelating peptides from rapeseed meal protein hydrolysates: identification and evaluation of their capacity to inhibit lipid oxidation.

An optimized proteolysis process was applied to rapeseed meal proteins (RP) and the hydrolysate was separated by membrane filtration allowing the production of highly metal-chelating peptides in the permeate. In order to identify the chemical structure of the most active obtained metal-chelating peptides, immobilized metal affinity chromatography (IMAC) was applied. The RP-IMAC peptide fraction was mainly composed of small peptides from 2 to 20 amino acids. Using the Ferrozine assay, RP-IMAC peptides showed a significant chelating efficiency higher than sodium citrate and close to that of EDTA. The peptide sequences were identified by UHPLC-MS and several possible iron binding sites were found. β-carotene oxidation assay and lipid oxidation in bulk oils or emulsion were carried out to evaluate the potential of such peptides as efficient antioxidants to protect lipids from oxidation. While chelating peptides showed a limited efficiency in bulk oil, they performed more efficiently in emulsion.