Targeting the proteolytic processing of the viral glycoprotein precursor is a promising novel antiviral strategy against arenaviruses.

A crucial step in the arenavirus life cycle is the biosynthesis of the viral envelope glycoprotein (GP) responsible for virus attachment and entry. Processing of the GP precursor (GPC) by the cellular proprotein convertase site 1 protease (S1P), also known as subtilisin-kexin-isozyme 1 (SKI-1), is crucial for cell-to-cell propagation of infection and production of infectious virus. Here, we sought to evaluate arenavirus GPC processing by S1P as a target for antiviral therapy using a recently developed peptide-based S1P inhibitor, decanoyl (dec)-RRLL-chloromethylketone (CMK), and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV). To control for off-target effects of dec-RRLL-CMK, we employed arenavirus reverse genetics to introduce a furin recognition site into the GPC of LCMV. The rescued mutant virus grew to normal titers, and the processing of its GPC critically depended on cellular furin, but not S1P. Treatment with the S1P inhibitor dec-RRLL-CMK resulted in specific blocking of viral spread and virus production of LCMV. Combination of the protease inhibitor with ribavirin, currently used clinically for treatment of human arenavirus infections, resulted in additive drug effects. In cells deficient in S1P, the furin-dependent LCMV variant established persistent infection, whereas wild-type LCMV underwent extinction without the emergence of S1P-independent escape variants. Together, the potent antiviral activity of an inhibitor of S1P-dependent GPC cleavage, the additive antiviral effect with ribavirin, and the low probability of emergence of S1P-independent viral escape variants make S1P-mediated GPC processing by peptide-derived inhibitors a promising strategy for the development of novel antiarenaviral drugs.

Amyloid Beta Precursor Protein: Proper Credit for the Basic Biochemical Properties of the Most Studied Protein in the 21st Century

The amyloid precursor protein (APP) is mainly known for being the precursor of the ß-amyloid peptide, which accumulates in plaques found in the brain of Alzheimer's disease patients. Expression in different tissues and the degree of sequence identity among mammals indicate an essential and non-tissue specific physiological function. APP is anchored to the membrane and displays a single C-terminal intracellular domain and a longer N-terminal extracellular domain. The basic biochemical properties and the scattered data on research, not related to production of beta-amyloid peptide, suggest that the protein and the molecules resulting from APP proteolytic cleavage may act as adhesion factors, enzymes, hormones/neurotransmitters and/or protease inhibitors. APP deserves to be known for its quite notable properties and its physiological role(s).

Molecular interaction of the human metalloprotease meprin beta with the amyloid precursor protein, ADAM10, and ADAM17 based on proteomics

Die Zinkendopeptidasen Meprin α und β sind Schlüsselkomponenten in patho(physiologischen) Prozessen wie Entzündung, Kollagenassemblierung und Angiogenese. Nach ihrer Entdeckung in murinen Bürstensaummembranen und humanen Darmepithelien, wurden weitere Expressionsorte identifiziert, z.B. Leukozyten, Krebszellen und die humane Haut. Tiermodelle, Zellkulturen und biochemische Analysen weisen auf Funktionen der Meprine in der Epithelialdifferenzierung, Zellmigration, Matrixmodellierung, Angiogenese, Bindegewebsausbildung und immunologische Prozesse hin. Dennoch sind ihre physiologischen Substrate weitgehend noch unbekannt. Massenspektrometrisch basierte Proteomics-Analysen enthüllten eine einzigartige Spaltspezifität für saure Aminosäurereste in der P1´ Position und identifizierten neue biologische Substratkandidaten. Unter den 269 extrazellulären Proteinen, die in einem Substratscreen identifiziert wurden, stellten sich das amyloid precursor protein (APP) and ADAM10 (a disintegrin and metalloprotease 10) als sehr vielversprechende Kandidaten heraus. Mehrere Schnittstellen innerhalb des APP Proteins, hervorgerufen durch verschiedenen Proteasen, haben unterschiedlichen Auswirkungen zur Folge. Die β-Sekretase BACE (β-site APP cleaving enzyme) prozessiert APP an einer Schnittstelle, welche als initialer Schritt in der Entwicklung der Alzheimer Erkrankung gilt. Toxische Aβ (Amyloid β)-Peptide werden in den extrazellulären Raum freigesetzt und aggregieren dort zu senilen Plaques. Membran verankertes Meprin β hat eine β-Sekretase Aktivität, die in einem Zellkultur-basierten System bestätigt werden konnte. Die proteolytische Effizienz von Meprin β wurde in FRET (Fluorescence Resonance Energy Transfer)-Analysen bestimmt und war um den Faktor 104 höher als die von BACE1. Weiterhin konnte gezeigt werden, dass Meprin β die ersten zwei Aminosäuren prozessiert und somit aminoterminal einen Glutamatrest freisetzt, welcher nachfolgend durch die Glutaminylzyklase in ein Pyroglutamat zykliert werden kann. Trunkierte Aβ-Peptide werden nur in Alzheimer Patienten generiert. Aufgrund einer erhöhten Hydrophobie weisen diese Peptide eine höhere Tendenz zur Aggregation auf und somit eine erhöhte Toxizität. Bis heute wurde keine Protease identifiziert, welche diese Schnittstelle prozessiert. Die Bildung der Meprin vermittelten N-terminalen APP Fragmenten wurde in vitro und in vivo detektiert. Diese N-APP Peptide hatten keine cytotoxischen Auswirkungen auf murine und humane Gehirnzellen, obwohl zuvor N-APP als Ligand für den death receptor (DR) 6 identifiziert wurde, der für axonale Degenerationsprozesse verantwortlich ist. rnIm nicht-amyloidogenen Weg prozessiert ADAM10 APP und entlässt die Ektodomäne von der Zellmembran. Wir konnten das ADAM10 Propeptid als Substrat von Meprin β identifizieren und in FRET Analysen, in vitro und in vivo zeigen, dass die Meprin vermittelte Prozessierung zu einer erhöhten ADAM10 Aktivität führt. Darüber hinaus wurde ADAM10 als Sheddase für Meprin β identifiziert. Shedding konnte durch Phorbol 12-myristate 13-acetate (PMA) oder durch das Ionophor A23187 hervorgerufen werden, sowie durch ADAM10 Inhibitoren blockiert werden. rnDiese Arbeit konnte somit ein komplexes proteolytisches Netwerk innerhalb der Neurophysiologie aufdecken, welches für die Entwicklung der Alzheimer Demenz wichtig sein kann.rn

Dimerization of β-site β-amyloid precursor protein-cleaving enzyme

Cleavage of the beta-amyloid precursor protein (APP) by the aspartyl protease beta-site APP-cleaving enzyme (BACE) is the first step in the generation of the amyloid beta-peptide, which is deposited in the brain of Alzheimer's disease patients. Whereas the subsequent cleavage by gamma-secretase was shown to originate from the cooperation of a multicomponent complex, it is currently unknown whether in a cellular environment BACE is enzymatically active as a monomer or in concert with other proteins. Using blue native gel electrophoresis we found that endogenous and overexpressed BACE has a molecular mass of 140 kDa instead of the expected mass of 70 kDa under denaturing conditions. This suggests that under native conditions BACE exists as a homodimer. Homodimerization was confirmed by co-immunoprecipitation of full-length BACE carrying different epitope tags. In contrast, the soluble active BACE ectodomain was exclusively present as a monomer both under native and denaturing conditions. A domain analysis revealed that the BACE ectodomain dimerized as long as it was attached to the membrane, whereas the cytoplasmic domain and the transmembrane domain were dispensable for dimerization. By adding a KKXX-endoplasmic reticulum retention signal to BACE, we demonstrate that dimerization of BACE occurs already before full maturation and pro-peptide cleavage. Furthermore, kinetic analysis of the purified native BACE dimer revealed a higher affinity and turnover rate in comparison to the monomeric soluble BACE. Dimerization of BACE might, thus, facilitate binding and cleavage of physiological substrates.

Evidence that the 42- and 40-amino acid forms of amyloid β protein are generated from the β-amyloid precursor protein by different protease activities

Cerebral deposition of the amyloid β protein (Aβ) is an early and invariant feature of Alzheimer disease (AD). Whereas the 40-amino acid form of Aβ (Aβ40) accounts for ≈90% of all Aβ normally released from cells, it appears to contribute only to later phases of the pathology. In contrast, the longer more amyloidogenic 42-residue form (Aβ42), accounting for only ≈10% of secreted Aβ, is deposited in the earliest phase of AD and remains the major constituent of most amyloid plaques throughout the disease. Moreover, its levels have been shown to be increased in all known forms of early-onset familial AD. Thus, inhibition of Aβ42 production is a prime therapeutic goal. The same protease, γ-secretase, is assumed to generate the C termini of both Aβ40 and Aβ42. Herein, we analyze the effect of the compound MDL 28170, previously suggested to inhibit γ-secretase, on β-amyloid precursor protein processing. By immunoprecipitating conditioned medium of different cell lines with various Aβ40- and Aβ42-specific antibodies, we demonstrate a much stronger inhibition of the γ-secretase cleavage at residue 40 than of that at residue 42. These data suggest that different proteases generate the Aβ40 and Aβ42 C termini. Further, they raise the possibility of identifying compounds that do not interfere with general β-amyloid precursor protein metabolism, including Aβ40 production, but specifically block the generation of the pathogenic Aβ42 peptide.

Three small nucleolar RNAs that are involved in ribosomal RNA precursor processing

Three small nucleolar RNAs (snoRNAs), E1, E2 and E3, have been described that have unique sequences and interact directly with unique segments of pre-rRNA in vivo. In this report, injection of antisense oligodeoxynucleotides into Xenopus laevis oocytes was used to target the specific degradation of these snoRNAs. Specific disruptions of pre-rRNA processing were then observed, which were reversed by injection of the corresponding in vitro-synthesized snoRNA. Degradation of each of these three snoRNAs produced a unique rRNA maturation phenotype. E1 RNA depletion shut down 18 rRNA formation, without overaccumulation of 20S pre-rRNA. After E2 RNA degradation, production of 18S rRNA and 36S pre-rRNA stopped, and 38S pre-rRNA accumulated, without overaccumulation of 20S pre-rRNA. E3 RNA depletion induced the accumulation of 36S pre-rRNA. This suggests that each of these snoRNAs plays a different role in pre-rRNA processing and indicates that E1 and E2 RNAs are essential for 18S rRNA formation. The available data support the proposal that these snoRNAs are at least involved in pre-rRNA processing at the following pre-rRNA cleavage sites: E1 at the 5′ end and E2 at the 3′ end of 18S rRNA, and E3 at or near the 5′ end of 5.8S rRNA.

The 9-cis-epoxycarotenoid cleavage reaction is the key regulatory step of abscisic acid biosynthesis in water-stressed bean

Abscisic acid (ABA), a cleavage product of carotenoids, is involved in stress responses in plants. A well known response of plants to water stress is accumulation of ABA, which is caused by de novo synthesis. The limiting step of ABA biosynthesis in plants is presumably the cleavage of 9-cis-epoxycarotenoids, the first committed step of ABA biosynthesis. This step generates the C15 intermediate xanthoxin and C25-apocarotenoids. A cDNA, PvNCED1, was cloned from wilted bean (Phaseolus vulgaris L.) leaves. The 2,398-bp full-length PvNCED1 has an ORF of 615 aa and encodes a 68-kDa protein. The PvNCED1 protein is imported into chloroplasts, where it is associated with the thylakoids. The recombinant protein PvNCED1 catalyzes the cleavage of 9-cis-violaxanthin and 9′-cis-neoxanthin, so that the enzyme is referred to as 9-cis-epoxycarotenoid dioxygenase. When detached bean leaves were water stressed, ABA accumulation was preceded by large increases in PvNCED1 mRNA and protein levels. Conversely, rehydration of stressed leaves caused a rapid decrease in PvNCED1 mRNA, protein, and ABA levels. In bean roots, a similar correlation among PvNCED1 mRNA, protein, and ABA levels was observed. However, the ABA content was much less than in leaves, presumably because of the much smaller carotenoid precursor pool in roots than in leaves. At 7°C, PvNCED1 mRNA and ABA were slowly induced by water stress, but, at 2°C, neither accumulated. The results provide evidence that drought-induced ABA biosynthesis is regulated by the 9-cis-epoxycarotenoid cleavage reaction and that this reaction takes place in the thylakoids, where the carotenoid substrate is located.

Human aspartic protease memapsin 2 cleaves the β-secretase site of β-amyloid precursor protein

The cDNAs of two new human membrane-associated aspartic proteases, memapsin 1 and memapsin 2, have been cloned and sequenced. The deduced amino acid sequences show that each contains the typical pre, pro, and aspartic protease regions, but each also has a C-terminal extension of over 80 residues, which includes a single transmembrane domain and a C-terminal cytosolic domain. Memapsin 2 mRNA is abundant in human brain. The protease domain of memapsin 2 cDNA was expressed in Escherichia coli and was purified. Recombinant memapsin 2 specifically hydrolyzed peptides derived from the β-secretase site of both the wild-type and Swedish mutant β-amyloid precursor protein (APP) with over 60-fold increase of catalytic efficiency for the latter. Expression of APP and memapsin 2 in HeLa cells showed that memapsin 2 cleaved the β-secretase site of APP intracellularly. These and other results suggest that memapsin 2 fits all of the criteria of β-secretase, which catalyzes the rate-limiting step of the in vivo production of the β-amyloid (Aβ) peptide leading to the progression of Alzheimer's disease. Recombinant memapsin 2 also cleaved a peptide derived from the processing site of presenilin 1, albeit with poor kinetic efficiency. Alignment of cleavage site sequences of peptides indicates that the specificity of memapsin 2 resides mainly at the S1′ subsite, which prefers small side chains such as Ala, Ser, and Asp.

Different cleavage sites are aligned differently in the active site of M1 RNA, the catalytic subunit of Escherichia coli RNase P.

We have studied RNase P RNA (M1 RNA) cleavage of model tRNA precursors that are cleaved at two independent positions. Here we present data demonstrating that cleavage at both sites depends on the 2'-OH immediately 5' of the respective cleavage site. However, we show that the 2-amino group of a guanosine at the cleavage site plays a significant role in cleavage at one of these sites but not at the other. These data suggest that these two cleavage sites are handled differently by the ribozyme. This theory is supported by our finding that the cross-linking pattern between Ml RNA and tRNA precursors carrying 4-thioU showed distinct differences, depending on the location of the 4-thioU relative to the respective cleavage site. These findings lead us to suggest that different cleavage sites are aligned differently in the active site, possibly as a result of different binding modes of a substrate to M1 RNA. We discuss a model in which the interaction between the 3'-terminal "RCCA" motif (first three residues interact) of a tRNA precursor and M1 RNA plays a significant role in this process.

Molecular cloning of a preprohormone from sea anemones containing numerous copies of a metamorphosis-inducing neuropeptide: a likely role for dipeptidyl aminopeptidase in neuropeptide precursor processing.

Neuropeptides are an important group of hormones mediating or modulating neuronal communication. Neuropeptides are especially abundant in evolutionarily "old" nervous systems, such as those of cnidarians, the lowest animal group having a nervous system. Cnidarians often have a life cycle including a polyp, a medusa, and a planula larva stage. Recently, a neuropeptide, < Glu-Gln-Pro-Gly-Leu-Trp-NH2, has been isolated from sea anemones that induces metamorphosis in a hydroid planula larva to become a hydropolyp [Leitz, T., Morand, K. & Mann, M. (1994) Dev. Biol. 163, 440-446]. Here, we have cloned the precursor protein for this metamorphosis-inducing neuropeptide from sea anemones. The precursor protein is 514-amino acid residues long and contains 10 copies of the immature, authentic neuropeptide (Gln-Gln-Pro-Gly-Leu-Trp-Gly). All neuropeptide copies are preceded by Xaa-Pro or Xaa-Ala sequences, suggesting a role for dipeptidyl aminopeptidase in neuropeptide precursor processing. In addition to these neuropeptide copies, there are 14 copies of another, closely related neuropeptide sequence (Gln-Asn-Pro-Gly-Leu-Trp-Gly). These copies are flanked by basic cleavage sites and, therefore, are likely to be released from the precursor protein. Furthermore, there are 13 other, related neuropeptide sequences having only small sequence variations (the most frequent sequence: Gln-Pro-Gly-Leu-Trp-Gly, eight copies). These variants are preceded by Lys-Arg, Xaa-Ala, or Xaa-Pro sequences, and are followed by basic cleavage sites, and therefore, are also likely to be produced from the precursor. Thus, there are at least 37 closely related neuropeptides localized on the precursor protein, making this precursor one of the most productive preprohormones known so far. This report also shows that unusual processing sites are common in cnidarian preprohormones.

Discovery, structural determination, and putative processing of the precursor protein that produces the cyclic trypsin inhibitor sunflower trypsin inhibitor 1

Backbone-cyclized proteins are becoming increasingly well known, although the mechanism by which they are processed from linear precursors is poorly understood. In this report the sequence and structure of the linear precursor of a cyclic trypsin inhibitor, sunflower trypsin inhibitor 1 (SFTI-1) from sunflower seeds, is described. The structure indicates that the major elements of the reactive site loop of SFTI-1 are present before processing. This may have importance for a protease-mediated cyclizing reaction as the rigidity of SFTI-1 may drive the equilibrium of the reaction catalyzed by proteolytic enzymes toward the formation of a peptide bond rather than the normal cleavage reaction. The occurrence of residues in the SFTI-1 precursor susceptible to cleavage by asparaginyl proteases strengthens theories that involve this enzyme in the processing of SFTI-1 and further implicates it in the processing of another family of plant cyclic proteins, the cyclotides. The precursor reported here also indicates that despite strong active site sequence homology, SFTI-1 has no other similarities with the Bowman-Birk trypsin inhibitors, presenting interesting evolutionary questions.

A proteo-liposome system for the analysis of the intracellular interactome of membrane proteins using amyloid precursor protein as a model

Transmembrane proteins play crucial roles in many important physiological processes. The intracellular domain of membrane proteins is key for their function by interacting with a wide variety of cytosolic proteins. It is therefore important to examine this interaction. A recently developed method to study these interactions, based on the use of liposomes as a model membrane, involves the covalent coupling of the cytoplasmic domains of membrane proteins to the liposome membrane. This allows for the analysis of interaction partners requiring both protein and membrane lipid binding. This thesis further establishes the liposome recruitment system and utilises it to examine the intracellular interactome of the amyloid precursor protein (APP), most well-known for its proteolytic cleavage that results in the production and accumulation of amyloid beta fragments, the main constituent of amyloid plaques in Alzheimer’s disease pathology. Despite this, the physiological function of APP remains largely unclear. Through the use of the proteo-liposome recruitment system two novel interactions of APP’s intracellular domain (AICD) are examined with a view to gaining a greater insight into APP’s physiological function. One of these novel interactions is between AICD and the mTOR complex, a serine/threonine protein kinase that integrates signals from nutrients and growth factors. The kinase domain of mTOR directly binds to AICD and the N-terminal amino acids of AICD are crucial for this interaction. The second novel interaction is between AICD and the endosomal PIKfyve complex, a lipid kinase involved in the production of phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) from phosphatidylinositol-3-phosphate, which has a role in controlling ensdosome dynamics. The scaffold protein Vac14 of the PIKfyve complex binds directly to AICD and the C-terminus of AICD is important for its interaction with the PIKfyve complex. Using a recently developed intracellular PI(3,5)P2 probe it is shown that APP controls the formation of PI(3,5)P2 positive vesicular structures and that the PIKfyve complex is involved in the trafficking and degradation of APP. Both of these novel APP interactors have important implications of both APP function and Alzheimer’s disease. The proteo-liposome recruitment method is further validated through its use to examine the recruitment and assembly of the AP-2/clathrin coat from purified components to two membrane proteins containing different sorting motifs. Taken together this thesis highlights the proteo-liposome recruitment system as a valuable tool for the study of membrane proteins intracellular interactome. It allows for the mimicking of the protein in its native configuration therefore identifying weaker interactions that are not detected by more conventional methods and also detecting interactions that are mediated by membrane phospholipids.

Tetraspanin 3: A central endocytic membrane component regulating the expression of ADAM10, presenilin and the amyloid precursor protein

Despite existing knowledge about the role of the A Disintegrin and Metalloproteinase 10 (ADAM10) as the α-secretase involved in the non-amyloidogenic processing of the amyloid precursor protein (APP) and Notch signalling we have only limited information about its regulation. In this study, we have identified ADAM10 interactors using a split ubiquitin yeast two hybrid approach. Tetraspanin 3 (Tspan3), which is highly expressed in the murine brain and elevated in brains of Alzheimer's disease (AD) patients, was identified and confirmed to bind ADAM10 by co-immunoprecipitation experiments in mammalian cells in complex with APP and the γ-secretase protease presenilin. Tspan3 expression increased the cell surface levels of its interacting partners and was mainly localized in early and late endosomes. In contrast to the previously described ADAM10-binding tetraspanins, Tspan3 did not affect the endoplasmic reticulum to plasma membrane transport of ADAM10. Heterologous Tspan3 expression significantly increased the appearance of carboxy-terminal cleavage products of ADAM10 and APP, whereas N-cadherin ectodomain shedding appeared unaffected. Inhibiting the endocytosis of Tspan3 by mutating a critical cytoplasmic tyrosine-based internalization motif led to increased surface expression of APP and ADAM10. After its downregulation in neuroblastoma cells and in brains of Tspan3-deficient mice, ADAM10 and APP levels appeared unaltered possibly due to a compensatory increase in the expression of Tspans 5 and 7, respectively. In conclusion, our data suggest that Tspan3 acts in concert with other tetraspanins as a stabilizing factor of active ADAM10, APP and the γ-secretase complex at the plasma membrane and within the endocytic pathway.

Zinc deficiency induces apoptosis via mitochondrial p53- and caspase-dependent pathways in human neuronal precursor cells

Previous studies have shown that zinc deficiency leads to apoptosis of neuronal precursor cells in vivo and in vitro. In addition to the role of p53 as a nuclear transcription factor in zinc deficient cultured human neuronal precursors (NT-2), we have now identified the translocation of phosphorylated p53 to the mitochondria and p53-dependent increases in the pro-apoptotic mitochondrial protein BAX leading to a loss of mitochondrial membrane potential as demonstrated by a 25% decrease in JC-1 red:green fluorescence ratio. Disruption of mitochondrial membrane integrity was accompanied by efflux of the apoptosis inducing factor (AIF) from the mitochondria and translocation to the nucleus with a significant increase in reactive oxygen species (ROS) after 24 h of zinc deficiency. Measurement of caspase cleavage, mRNA, and treatment with caspase inhibitors revealed the involvement of caspases 2, 3, 6, and 7 in zinc deficiency-mediated apoptosis. Down-stream targets of caspase activation, including the nuclear structure protein lamin and polyADP ribose polymerase (PARP), which participates in DNA repair, were also cleaved. Transfection with a dominant-negative p53 construct and use of the p53 inhibitor, pifithrin- ␮, established that these alterations were largely dependent on p53. Together these data identify a cascade of events involving mitochondrial p53 as well as p53-dependent caspase-mediated mechanisms leading to apoptosis during zinc deficiency.

Antitumor Activity Of Irradiated Riboflavin On Human Renal Carcinoma Cell Line 786-o.

Riboflavin (vitamin B2) is a precursor for coenzymes involved in energy production, biosynthesis, detoxification, and electron scavenging. Previously, we demonstrated that irradiated riboflavin (IR) has potential antitumoral effects against human leukemia cells (HL60), human prostate cancer cells (PC3), and mouse melanoma cells (B16F10) through a common mechanism that leads to apoptosis. Hence, we here investigated the effect of IR on 786-O cells, a known model cell line for clear cell renal cell carcinoma (CCRCC), which is characterized by high-risk metastasis and chemotherapy resistance. IR also induced cell death in 786-O cells by apoptosis, which was not prevented by antioxidant agents. IR treatment was characterized by downregulation of Fas ligand (TNF superfamily, member 6)/Fas (TNF receptor superfamily member 6) (FasL/Fas) and tumor necrosis factor receptor superfamily, member 1a (TNFR1)/TNFRSF1A-associated via death domain (TRADD)/TNF receptor-associated factor 2 (TRAF) signaling pathways (the extrinsic apoptosis pathway), while the intrinsic apoptotic pathway was upregulated, as observed by an elevated Bcl-2 associated x protein/B-cell CLL/lymphoma 2 (Bax/Bcl-2) ratio, reduced cellular inhibitor of apoptosis 1 (c-IAP1) expression, and increased expression of apoptosis-inducing factor (AIF). The observed cell death was caspase-dependent as proven by caspase 3 activation and poly(ADP-ribose) polymerase-1 (PARP) cleavage. IR-induced cell death was also associated with downregulation of v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homologue (avian)/protein serine/threonine kinase B/extracellular signal-regulated protein kinase 1/2 (Src/AKT/ERK1/2) pathway and activation of p38 MAP kinase (p38) and Jun-amino-terminal kinase (JNK). Interestingly, IR treatment leads to inhibition of matrix metalloproteinase-2 (MMP-2) activity and reduced expression of renal cancer aggressiveness markers caveolin-1, low molecular weight phosphotyrosine protein phosphatase (LMWPTP), and kinase insert domain receptor (a type III receptor tyrosine kinase) (VEGFR-2). Together, these results show the potential of IR for treating cancer.